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Azurin is a small periplasmic blue copper protein found in bacterial strains such as Pseudomonas and Alcaligenes where it facilitates denitrification. Azurin is extensively studied for its ability to mediate electron-transfer processes, but it has also sparked interest of the pharmaceutical community as a potential antimicrobial or anticancer agent. Here we offer a novel approach for expression and single-step purification of azurin in Escherichia coli with high yields and optimal metalation. A fusion tag strategy using an N-terminal GST tag was employed to obtain pure protein without requiring any additional purification steps. After the on-column cleavage by HRV 3C Protease, azurin is collected and additionally incubated with copper sulphate to ensure sufficient metalation. UV-VIS absorption, mass spectroscopy, and circular dichroism analysis all validated the effective production of azurin, appropriate protein folding and the development of an active site with an associated cofactor. MD simulations verified that incorporation of the N-terminal GPLGS segment does not affect azurin structure. In addition, the biological activity of azurin was tested in HeLa cells., Competing Interests: Declaration of competing interest The authors declare no competing interests., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Azurin, a secondary metabolite from Pseudomonas aeruginosa, has attracted much attention owing to its valuable therapeutic and biological applications. This work aimed to study and chartly maximize the azurin production process using different doses of gamma irradiation (5-400 Gy) in P. aeruginosa isolates. Seventy-six P. aeruginosa isolates were sourced from 135 environmental samples and 35 clinical bacterial isolates with the following descending order: 35 isolates (46%) from clinical samples, 26 isolates (34%) from water samples, and 15 isolates (20%) from soil samples. The disc diffusion technique was used for antimicrobial susceptibility testing, revealing that the multidrug-resistant (MDR) rate among all collected isolates according to the criteria determined by Clinical and Laboratory Standards Institute (CLSI) was 54 (71%). The genomic experimental results revealed that only 37 MDR isolates tested positive for the azurin gene, as detected by the PCR product at 446 bp. These findings were further supported by FTIR analysis, which revealed peaks around 1636.96 cm - 1 , indicating a prominent α-helix secondary structure of azurin in these isolates. Related to their pathogenicity and antibiotic resistance, isolates from clinical origin exhibited the higher azurin gene expression level. Besides, this study confirmed the potency of gamma radiation exposure at 50 and 100 Gy significantly increased the azurin expression levels in three tested clinical isolates (P ≤ 0.05), with a maximum fold expression level of 63.55 compared to the non-irradiated samples. In conclusion, low doses of gamma irradiation effectively enhanced expression level of a secondary metabolite azurin, providing a considerable benefit for subsequent purification processes in both biological and medical applications., Competing Interests: Declarations. Ethical approval: Not applicable. Research involving human participation and/or animals: Not applicable. Informed consent: Not applicable. Competing interests: The authors declare no competing interests., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Copper-containing proteins play crucial roles in biological systems. Azurin is a copper-containing protein which has a Type 1 copper site that facilitates electron transfer in the cytochrome chain. Previous research has highlighted the significant impact of mutations in the axial Met121 of the copper site on the reduction potential. However, the mechanism of this regulation has not been fully established. In this study, we employed theoretical modeling to investigate the reduction of the Type 1 copper site, focusing on how unnatural amino acid substitutions at Met121 influence its behavior. Our findings demonstrated a strong linear correlation between electrostatic interactions and the reduction potential of the copper site, which indicates that the perturbation of the reduction potential is primarily influenced by electrostatic interactions between the metal ion and the ligating atom. Furthermore, we found that CF/π and CF…H interactions could induce subtle changes in geometry and hence impact the electronic properties of the systems under study. In addition, our calculations suggest the coordination mode and ion-ligand distance could significantly impact the reduction potential of a copper site. Overall, this study offers valuable insights into the structural and electronic properties of the Type 1 copper site, which could potentially guide the design of future artificial catalysts., Competing Interests: Declaration of competing interest The authors declare no competing financial interest., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Motivated by the ambition to establish an enzyme-driven bioleaching pathway for copper extraction, properties of the Type-1 copper protein rusticyanin from Acidithiobacillus ferrooxidans (AfR) were compared with those from an ancestral form of this enzyme (N0) and an archaeal enzyme identified in Ferroplasma acidiphilum (FaR). While both N0 and FaR show redox potentials similar to that of AfR their electron transport rates were significantly slower. The lack of a correlation between the redox potentials and electron transfer rates indicates that AfR and its associated electron transfer chain evolved to specifically facilitate the efficient conversion of the energy of iron oxidation to ATP formation. In F. acidiphilum this pathway is not as efficient unless it is up-regulated by an as of yet unknown mechanism. In addition, while the electrochemical properties of AfR were consistent with previous data, previously unreported behavior was found leading to a form that is associated with a partially unfolded form of the protein. The cyclic voltammetry (CV) response of AfR immobilized onto an electrode showed limited stability, which may be connected to the presence of the partially unfolded state of this protein. Insights gained in this study may thus inform the engineering of optimized rusticyanin variants for bioleaching processes as well as enzyme-catalyzed solubilization of copper-containing ores such as chalcopyrite., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023. Published by Elsevier Inc.)

Transfection efficiency of the immortalized human breast epithelial cell line MCF-10A remains an issue that needs to be resolved. In this study, it was aimed to deliver a recombinant DNA (pCMV-Azu-GFP) to the MCF-10A cells by the magnetofection method using magnetic nanoparticles (MNPs) and a simple magnet to accelerate the DNA delivery. Surface positively modified silica-coated iron oxide MNPs (MSNP-NH 2 ) were produced and characterized via TEM, FTIR, and DLS analyses. The recombinant DNA (rDNA) was obtained by the integration of codon-optimized azurin to produce a fusion protein. Then, rDNA cloned in Escherichia coli cells was validated by sequence analysis. The electrostatically conjugated rDNA on MSNP-NH 2 with an enhancer polyethyleneimine (PEI) was studied by agarose gel electrophoresis and the optimum conditions were determined to apply to the cell. A dose-dependent statistical difference was observed on treated cells based on the MTS test. The expression of the fusion protein after magnetofection was determined using laser scanning confocal microscope imaging and western blot analysis. It was observed that the azurin gene could be transferred to MCF-10A cells by magnetofection. Thus, when the azurin gene is used as a breast cancer treatment agent, it can be expressed in healthy cells without toxic effects., (© 2023. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Secondary coordination sphere (SCS) interactions have been shown to play important roles in tuning reduction potentials and electron transfer (ET) properties of the Type 1 copper proteins, but the precise roles of these interactions are not fully understood. In this work, we examined the influence of F114P, F114N, and N47S mutations in the SCS on the electronic structure of the T1 copper center in azurin (Az) by studying the hyperfine couplings of (i) histidine remote N ε nitrogens and (ii) the amide N p using the two-dimensional (2D) pulsed electron paramagnetic resonance (EPR) technique HYSCORE (hyperfine sublevel correlation) combined with quantum mechanics/molecular mechanics (QM/MM) and DLPNO-CCSD calculations. Our data show that some components of hyperfine tensor and isotropic coupling in N47SAz and F114PAz (but not F114NAz) deviate by up to ∼±20% from WTAz, indicating that these mutations significantly influence the spin density distribution between the Cu II site and coordinating ligands. Furthermore, our calculations support the assignment of N p to the backbone amide of residue 47 (both in Asn and Ser variants). Since the spin density distributions play an important role in tuning the covalency of the Cu-Scys bond of Type 1 copper center that has been shown to be crucial in controlling the reduction potentials, this study provides additional insights into the electron spin factor in tuning the reduction potentials and ET properties.

Cupredoxins are widely occurring copper-binding proteins with a typical Greek-key beta barrel fold. They are generally described as electron carriers that rely on a T1 copper centre coordinated by four ligands provided by the folded polypeptide. The discovery of novel cupredoxins demonstrates the high diversity of this family, with variations in terms of copper-binding ligands, copper centre geometry, redox potential, as well as biological function. AcoP is a periplasmic cupredoxin belonging to the iron respiratory chain of the acidophilic bacterium Acidithiobacillus ferrooxidans. AcoP presents original features, including high resistance to acidic pH and a constrained green-type copper centre of high redox potential. To understand the unique properties of AcoP, we undertook structural and biophysical characterization of wild-type AcoP and of two Cu-ligand mutants (H166A and M171A). The crystallographic structures, including native reduced AcoP at 1.65 Å resolution, unveil a typical cupredoxin fold. The presence of extended loops, never observed in previously characterized cupredoxins, might account for the interaction of AcoP with physiological partners. The Cu-ligand distances, determined by both X-ray diffraction and EXAFS, show that the AcoP metal centre seems to present both T1 and T1.5 features, in turn suggesting that AcoP might not fit well to the coupled distortion model. The crystal structures of two AcoP mutants confirm that the active centre of AcoP is highly constrained. Comparative analysis with other cupredoxins of known structures, suggests that in AcoP the second coordination sphere might be an important determinant of active centre rigidity due to the presence of an extensive hydrogen bond network. Finally, we show that other cupredoxins do not perfectly follow the coupled distortion model as well, raising the suspicion that further alternative models to describe copper centre geometries need to be developed, while the importance of rack-induced contributions should not be underestimated.

A fusion protein composed of a bacterial protein, azurin, having antineoplastic properties and a thermally responsive structural cationic elastin-like protein (ELP), is designed, cloned, expressed, and purified. A simple method of inverse transition cycle (ITC) is employed to purify the fusion protein azurin-ELP diblock copolymer (d-bc). The molecular weight of the azurin-ELP fusion protein is ∼32 kDa. Further, its self-assembly properties are investigated. Interestingly, the engineered azurin-ELP d-bc in response to increasing temperature shows a dual-step phase separation into biofunctional nanostructures. Around the physiological temperature, azurin-ELP d-bc forms stable coacervates, which is dependent on the concentration and time of incubation. These coacervates are formed below the lower critical solubility temperature (LCST) of the ELP block at physiological temperature. Above LCST, i.e., 50-55°C, micelles of size ranging from 25 to 30 nm are formed. The cytotoxicity of azurin-ELP d-bc depends on the size of the coacervates formed and their cellular uptake at physiological temperature. Further, MTT assay of azurin-ELP d-bc in the cross-linked micelles prepared ex situ shows > six times higher killing of LNCaP cells than the unimeric form of azurin-ELP at 5 μM concentration. The flow cytometric results of these micelles at 20 μM concentration show ∼97% LNCaP cells in the apoptotic phase. Thus, azurin-ELP cross-linked micelles have enhanced potential for anticancer therapy due to their higher avidity.

Erythropoietin-producing hepatocellular (Eph) receptors and their ligands, ephrins, are the largest subfamily of receptor tyrosine kinases (RTKs) that have emerged as a new class of cancer biomarkers due to their aberrant expression in cancer progression. The activation of Eph receptors either due to their hyperexpression or via high affinity binding with their respective ephrin ligands initiates a cascade of signals that impacts cancer development and progression. In prostate cancer, the overexpression of the EphA6 receptor has been correlated with increased metastatic potential. Azurin, a small redox protein, is known to prevent tumor progression by binding to cell surface Eph receptors, inhibiting its autophosphorylation in the kinase domain and thereby disrupting Eph-ephrin signaling. Hence, a self-assembled, theranostic nanosystem of recombinant fusion protein his 6 EGFP-azu (80-128) was designed by conjugating enhanced green fluorescent protein (EGFP) with the C-terminal region of azurin. This design was inspired by the in silico binding study, where the analogue of ephrinA, his 6 EGFP-azu (80-128) showed higher binding affinity for the EphA6 receptor than the ephrinA ligands. The his 6 EGFP-azu (80-128) nanosystem which assembled as nanoparticles was tested for its ability to simultaneously detect and kill the prostate cancer cells, LNCaP. This was achieved by specifically targeting EphA6 receptors overexpressed on the cancer cell surface via C-terminal peptide, azu (80-128). Herein, we report antiproliferative, apoptotic, antimigratory, and anti-invasive effects of this nanosystem on LNCaP cells, while having no similar effects on EphA6 negative human normal lung cells, WI-38.

Circular permutation (CP) is a technique by which the primary sequence of a protein is rearranged to create new termini. The connectivity of the protein is altered but the overall protein structure generally remains unperturbed. Understanding the effect of CP can help design robust proteins for numerous applications such as in genetic engineering, optoelectronics, and improving catalytic activity. Studies on different protein topologies showed that CP usually affects protein stability as well as unfolding rates. Though a significant number of proteins contain metals or other cofactors, reports of metalloprotein CPs are rare. Thus, we chose a bacterial metalloprotein, azurin, and its CP within the metal-binding site (cpF114). We studied the stabilities, folding, and unfolding rates of apo- and Zn 2+ -bound CP azurin using fluorescence and circular dichroism. The introduced CP had destabilizing effects on the protein. Also, the folding of the Zn 2+ -CP protein was much slower than that of the Zn 2+ -WT or apo-protein. We compared this study to our previously reported azurin-cpN42, where we had observed an equilibrium and kinetic intermediate. cpF114 exhibits an apparent two-state equilibrium unfolding but has an off-pathway kinetic intermediate. Our study hinted at CP as a method to modify the energy landscape of proteins to alter their folding pathways. WT azurin, being a faster folder, may have evolved to optimize the folding rate of metal-bound protein compared to its CPs, albeit all of them have the same structure and function. Our study underscores that protein sequence and protein termini positions are crucial for metalloproteins. TOC Figure. (Top) Zn 2+ -azurin WT structure (PDB code: 1E67) and 2-D topology diagram of Zn 2+ -cpF114 azurin. (Bottom) Cartoon diagram representing folding (red arrows) and unfolding (blue arrows) of apo- and Zn 2+ - WT and cpF114 azurins. The width of the arrows represents the rate of the corresponding processes., (© 2023. The Author(s), under exclusive licence to Society for Biological Inorganic Chemistry (SBIC).)

Tryptophan serves as an important redox-active amino acid in mediating electron transfer and mitigating oxidative damage in proteins. We previously showed a difference in electrochemical potentials for two tryptophan residues in azurin with distinct hydrogen-bonding environments. Here, we test whether reducing the side chain bulk at position Phe110 to Leu, Ser, or Ala impacts the electrochemical potentials ( E °) for tryptophan at position 48. X-ray diffraction confirmed the influx of crystallographically resolved water molecules for both the F110A and F110L tyrosine free azurin mutants. The local environments of W48 in all azurin mutants were further evaluated by UV resonance Raman (UVRR) spectroscopy to probe the impact of mutations on hydrogen bonding and polarity. A correlation between the frequency of the ω17 mode─considered a vibrational marker for hydrogen bonding─and E ° is proposed. However, the trend is opposite to the expectation from a previous study on small molecules. Density functional theory calculations suggest that the ω17 mode reflects hydrogen bonding as well as local polarity. Further, the UVRR data reveal different intensity/frequency shifts of the ω9/ω10 vibrational modes that characterize the local H-bonding environments of tryptophan. The cumulative data support that the presence of water increases E ° and reveal properties of the protein microenvironment surrounding tryptophan.

Microorganisms living at many sites in the human body compose a complex and dynamic community. Accumulating evidence suggests a significant role for microorganisms in cancer, and therapies that incorporate bacteria have been tried in various types of cancer. We previously demonstrated that cupredoxin azurin secreted by the opportunistic pathogen Pseudomonas aeruginosa, enters human cancer cells and induces apoptotic death 1-4 . However, the physiological interactions between P. aeruginosa and humans and their role in tumor homeostasis are largely unknown. Here, we show that P. aeruginosa upregulated azurin secretion in response to increasing numbers of and proximity to cancer cells. Conversely, cancer cells upregulated aldolase A secretion in response to increasing proximity to P. aeruginosa, which also correlated with enhanced P. aeruginosa adherence to cancer cells. Additionally, we show that cancer patients had detectable P. aeruginosa and azurin in their tumors and exhibited increased overall survival when they did, and that azurin administration reduced tumor growth in transgenic mice. Our results suggest host-bacterial symbiotic mutualism acting as a diverse adjunct to the host defense system via inter-kingdom communication mediated by the evolutionarily conserved proteins azurin and human aldolase A. This improved understanding of the symbiotic relationship of bacteria with humans indicates the potential contribution to tumor homeostasis., (© 2023. The Author(s).)

Background: Azurin, a bacterial cupredoxin firstly isolated from the bacterium Pseudomonas aeruginosa , is considered a potential alternative therapeutic tool against different types of cancer., Aims: In this work we have explored the relationship possibly existing between azurin and colorectal cancer (CRC), in light of the evidence that microbial imbalance can lead to CRC progression., Methodology/results: To this aim, the presence of azurin coding gene in the DNA extracted from saliva, stool, and biopsy samples of 10 CRC patients and 10 healthy controls was evaluated by real-time PCR using primers specifically designed to target the azurin coding gene from different bacterial groups. The correlation of the previously obtained microbiota data with real-time PCR results evidenced a "preferential" enrichment of seven bacterial groups in some samples than in others, even though no statistical significance was detected between controls and CRC. The subset of azurin gene-harbouring bacterial groups was representative of the entire community., Conclusions: Despite the lack of statistical significance between healthy and diseased patients, HTS data analysis highlighted a kind of "preferential" enrichment of seven bacterial groups harbouring the azurin gene in some samples than in others., Competing Interests: The authors declare no conflict of interest. AA is serving as one of the Editorial Board members and Guest editors of this journal. We declare that AA had no involvement in the peer review of this article and has no access to information regarding its peer review. Full responsibility for the editorial process for this article was delegated to RC., (© 2022 The Author(s). Published by IMR Press.)

The protein, azurin, has enabled the study of the tryptophan radical. Upon UV excitation of tyrosine-deficient apoazurin and in the presence of a Co(III) electron acceptor, the neutral radical (W48•) is formed. The lifetime of W48• in apoazurin is 41 s, which is shorter than the lifetime of several hours in Zn-substituted azurin. Molecular dynamics simulations revealed enhanced fluctuations of apoazurin which likely destabilize W48•. The photophysics of W48 was investigated to probe the precursor state for ET. The phosphorescence intensity was eliminated in the presence of an electron acceptor while the fluorescence was unchanged; this quenching of the phosphorescence is attributed to ET. The kinetics associated with W48• were examined with a model that incorporates intersystem crossing, ET, deprotonation, and decay of the cation radical. The estimated rate constants for ET (6 × 10 6 s -1 ) and deprotonation (3 × 10 5 s -1 ) are in agreement with a photoinduced mechanism where W48• is derived from the triplet state. The triplet as the precursor state for ET was supported by photolysis of apoazurin with 280 nm in the absence and presence of triplet-absorbing 405 nm light. Absorption bands from the neutral radical were observed only in the presence of blue light.

The rusticyanin protein, a blue monomeric copper protein type-1, is one of the main components in the iron-electron transfer chain of the Acidithiobacillus ferrooxidans, and is the product of the rus gene expression. Herein, first the bacterial DNA of Acidithiobacillus sp. FJ2 was extracted. Then, the rus gene sequence and the sequence amino acid rusticyanin protein were determined. The Met148Leu mutation increased the oxidase activity of the rusticyanin protein, thereby enhancing the efficiency of the bioleaching process by bacteria Acidithiobacillus ferroxidans . Met148Leu mutation was created in the rusticyanin protein, then molecular dynamics (MD) simulations and structural analysis were performed. The MD analysis of the wild-type and mutant protein demonstrated a slight instability in the mutant protein and significant instability in the active site of the mutant protein. The usefulness of this study is the genetic manipulation of the native Acidithiobacillus sp. FJ2 bacterium, which can boost the bioleaching efficiency of the bacterium to some extent, and investigating its effects on the structure of a mutant protein using computational methods.

Hole hopping through tryptophan/tyrosine chains enables rapid unidirectional charge transport over long distances. We have elucidated structural and dynamical factors controlling hopping speed and efficiency in two modified azurin constructs that include a rhenium(I) sensitizer, Re(His)(CO) 3 (dmp) + , and one or two tryptophans (W 1 , W 2 ). Experimental kinetics investigations showed that the two closely spaced (3 to 4 Å) intervening tryptophans dramatically accelerated long-range electron transfer (ET) from Cu I to the photoexcited sensitizer. In our theoretical work, we found that time-dependent density-functional theory (TDDFT) quantum mechanics/molecular mechanics/molecular dynamics (QM/MM/MD) trajectories of low-lying triplet excited states of Re I (His)(CO) 3 (dmp) + -W 1 (-W 2 ) exhibited crossings between sensitizer-localized (*Re) and charge-separated [Re I (His)(CO) 3 (dmp •- )/(W 1 •+ or W 2 •+ )] (CS1 or CS2) states. Our analysis revealed that the distances, angles, and mutual orientations of ET-active cofactors fluctuate in a relatively narrow range in which the cofactors are strongly coupled, enabling adiabatic ET. Water-dominated electrostatic field fluctuations bring *Re and CS1 states to a crossing where *Re(CO) 3 (dmp) + ←W 1 ET occurs, and CS1 becomes the lowest triplet state. ET is promoted by solvation dynamics around *Re(CO) 3 (dmp) + (W 1 ); and CS1 is stabilized by Re(dmp •- )/W 1 •+ electron/hole interaction and enhanced W 1 •+ solvation. The second hop, W 1 •+ ←W 2 , is facilitated by water fluctuations near the W 1 /W 2 unit, taking place when the electrostatic potential at W 2 drops well below that at W 1 •+ Insufficient solvation and reorganization around W 2 make W 1 •+ ←W 2 ET endergonic, shifting the equilibrium toward W 1 •+ and decreasing the charge-separation yield. We suggest that multiscale TDDFT/MM/MD is a suitable technique to model the simultaneous evolution of photogenerated excited-state manifolds., Competing Interests: The authors declare no competing interest.

Cu-containing nitrite reductases that convert NO 2 - to NO are critical enzymes in nitrogen-based energy metabolism. Among organisms in the order Rhizobiales, we have identified two copies of nirK, one encoding a new class of 4-domain CuNiR that has both cytochrome and cupredoxin domains fused at the N terminus and the other, a classical 2-domain CuNiR (Br 2D NiR). We report the first enzymatic studies of a novel 4-domain CuNiR from Bradyrhizobium sp. ORS 375 (BrNiR), its genetically engineered 3- and 2-domain variants, and Br 2D NiR revealing up to ~ 500-fold difference in catalytic efficiency in comparison with classical 2-domain CuNiRs. Contrary to the expectation that tethering would enhance electron delivery by restricting the conformational search by having a self-contained donor-acceptor system, we demonstrate that 4-domain BrNiR utilizes N-terminal tethering for downregulating enzymatic activity instead. Both Br 2D NiR and an engineered 2-domain variant of BrNiR (Δ(Cytc-Cup) BrNiR) have 3 to 5% NiR activity compared to the well-characterized 2-domain CuNiRs from Alcaligenes xylosoxidans (AxNiR) and Achromobacter cycloclastes (AcNiR). Structural comparison of Δ(Cytc-Cup) BrNiR and Br 2D NiR with classical 2-domain AxNiR and AcNiR reveals structural differences of the proton transfer pathway that could be responsible for the lowering of activity. Our study provides insights into unique structural and functional characteristics of naturally occurring 4-domain CuNiR and its engineered 3- and 2-domain variants. The reverse protein engineering approach utilized here has shed light onto the broader question of the evolution of transient encounter complexes and tethered electron transfer complexes. ENZYME: Copper-containing nitrite reductase (CuNiR) (EC 1.7.2.1). DATABASE: The atomic coordinate and structure factor of Δ(Cytc-Cup) BrNiR and Br 2D NiR have been deposited in the Protein Data Bank (http://www.rcsb.org/) under the accession code 6THE and 6THF, respectively., (© 2020 The Authors. The FEBS Journal published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

Azurin, which is a bacterial secondary metabolite has been attracted as a potential anticancer agent in recent years because induced death of cancer cells and inhibited their growth. In this study, the production of azurin under the control of the alcohol oxidase promoter which is frequently used in the Pichia pastoris expression system was performed. The azurin gene amplified from Pseudomonas aeruginosa genomic DNA and inserted into the pPICZαA was cloned in Escherichia coli cells. Then, a linearized recombinant vector was transferred to the P. pastoris X-33 cells. Antibiotic resistance test and colony PCR were performed for the selection of multicopy transformants. Protein expression capacities of selected transformants were compared at the end of 48 h incubation. Both extracellular and intracellular protein expressions were observed in all of them by Western blot analysis. The relative expression levels of both intracellular and extracellular protein that belongs to the first clone were higher than the others. On the other hand, it was seen that the 4th clone had the highest protein secretion ability. The molecular mass of the extracellular azurin protein which is produced by recombinant clones was found to be about 20 kDa. This is the first report on azurin expression in P. pastoris .

We observe reversible, bias-induced switching of conductance via a blue copper protein azurin mutant, N42C Az , with a nearly 10-fold increase at | V | > 0.8 V than at lower bias. No such switching is found for wild-type azurin, WT Az , up to |1.2 V|, beyond which irreversible changes occur. The N42C Az mutant will, when positioned between electrodes in a solid-state Au-protein-Au junction, have an orientation opposite that of WT Az with respect to the electrodes. Current(s) via both proteins are temperature-independent, consistent with quantum mechanical tunneling as dominant transport mechanism. No noticeable difference is resolved between the two proteins in conductance and inelastic electron tunneling spectra at <|0.5 V| bias voltages. Switching behavior persists from 15 K up to room temperature. The conductance peak is consistent with the system switching in and out of resonance with the changing bias. With further input from UV photoemission measurements on Au-protein systems, these striking differences in conductance are rationalized by having the location of the Cu(II) coordination sphere in the N42C Az mutant, proximal to the (larger) substrate-electrode, to which the protein is chemically bound, while for the WT Az that coordination sphere is closest to the other Au electrode, with which only physical contact is made. Our results establish the key roles that a protein's orientation and binding nature to the electrodes play in determining the electron transport tunnel barrier.

Electron Paramagnetic Resonance (EPR) spectroscopy is a powerful method for unraveling structures and dynamics of biomolecules. Out of the EPR tool box, Pulsed Electron-Electron Double Resonance spectroscopy (PELDOR or DEER) enables one to resolve such structures by providing distances between spin centers on the nanometer scale. Most commonly, both spin centers are spin labels or one is a spin label and the other is a paramagnetic metal ion, cluster, amino acid or cofactor radical. Often, the translation of the measured distances into structures is complicated by the long and flexible linker connecting the spin center of the spin label with the biomolecule. Nowadays, this challenge is overcome by computational methods but the currently available approaches have a rather large mean error of roughly 2-3 Å. Here, the new GFN-FF general force-field is combined with the fully automated Conformer Rotamer Ensemble Search Tool (CREST) [P. Pracht et al., Phys. Chem. Chem. Phys., 2020, 22, 7169-7192] to generate conformer ensembles of the R1 side chain (methanthiosulfonate spin label (MTSL) covalently bound to a cysteine) in several cysteine mutants of azurin and T4 lysozyme. In order to determine the Cu2+-R1 and R1-R1 distance distributions, GFN-FF based MD simulations were carried out starting from the most probable R1 conformers found by CREST. The deviation between theory and experiment in mean inter-spin distances was 0.98 Å on average for the mutants of azurin (1.84 Å for T4 lysozyme) and the right modality was obtained. The error of the most probable distances for each mode was only 0.76 Å in the case of azurin. This CREST/MD procedure does thus enable precise distance-to-structure translations and provides a means to disentangle label from protein conformers.

Acidithiobacillus ferrooxidans cells can oxidize iron and sulfur and are key members of the microbial biomining communities that are exploited in the large-scale bioleaching of metal sulfide ores. Some minerals are recalcitrant to bioleaching due to the presence of other inhibitory materials in the ore bodies. Additives are intentionally included in processed metals to reduce environmental impacts and microbially influenced corrosion. We have previously reported a new aerobic corrosion mechanism where A. ferrooxidans cells combined with pyrite and chloride can oxidize low-grade stainless steel (SS304) with a thiosulfate-mediated mechanism. Here we explore process conditions and genetic engineering of the cells that enable corrosion of a higher grade steel (SS316). The addition of elemental sulfur and an increase in the cell loading resulted in a 74% increase in the corrosion of SS316 as compared to the initial sulfur- and cell-free control experiments containing only pyrite. The overexpression of the endogenous rus gene, which is involved in the cellular iron oxidation pathway, led to a further 85% increase in the corrosion of the steel in addition to the improvements made by changes to the process conditions. Thus, the modification of the culturing conditions and the use of rus-overexpressing cells led to a more than threefold increase in the corrosion of SS316 stainless steel, such that 15% of the metal coupons was dissolved in just 2 weeks. This study demonstrates how the engineering of cells and the optimization of their cultivation conditions can be used to discover conditions that lead to the corrosion of a complex metal target., (© 2020 Wiley Periodicals LLC.)

Time-resolved phosphorescence spectra of Re(CO) 3 (dmp) + and Re(CO) 3 (phen) + chromophores (dmp = 4,7-dimethyl-1,10-phenanthroline, phen = 1,10-phenanthroline) bound to surface histidines (H83, H124, and H126) of Pseudomonas aeruginosa azurin mutants exhibit dynamic band maxima shifts to lower wavenumbers following 3-exponential kinetics with 1-5 and 20-100 ns major phases and a 1.1-2.5 μs minor (5-16%) phase. Observation of slow relaxation components was made possible by using an organometallic Re chromophore as a probe whose long phosphorescence lifetime extends the observation window up to ∼3 μs. Integrated emission-band areas also decay with 2- or 3-exponential kinetics; the faster decay phase(s) is relaxation-related, whereas the slowest one [360-680 ns (dmp); 90-140 ns (phen)] arises mainly from population decay. As a result of shifting bands, the emission intensity decay kinetics depend on the detection wavelength. Detailed kinetics analyses and comparisons with band-shift dynamics are needed to disentangle relaxation and population decay kinetics if they occur on comparable timescales. The dynamic phosphorescence Stokes shift in Re-azurins is caused by relaxation motions of the solvent, the protein, and solvated amino acid side chains at the Re binding site in response to chromophore electronic excitation. Comparing relaxation and decay kinetics of Re(dmp)124K122Cu II and Re(dmp)124W122Cu II suggests that electron transfer (ET) and relaxation motions in the W122 mutant are coupled. It follows that nanosecond and faster photo-induced ET steps in azurins (and likely other redox proteins) occur from unrelaxed systems; importantly, these reactions can be driven (or hindered) by structural and solvational dynamics.

Azurin, a bacteriocin produced by a human gut bacterium Pseudomonas aeruginosa, can reveal selectively cytotoxic and induce apoptosis in cancer cells. After overcoming two phase I trials, a functional region of Azurin called p28 has been approved as a drug for the treatment of brain tumor glioma by FDA. The present study aims to improve a screening procedure and assess genetic diversity of Azurin genes in P. aeruginosa and Azurin-like genes in the gut microbiome of a specific population in Vietnam and global populations. Firstly, both cultivation-dependent and cultivation-independent techniques based on genomic and metagenomic DNAs extracted from fecal samples of the healthy specific population were performed and optimized to detect Azurin genes. Secondly, the Azurin gene sequences were analyzed and compared with global populations by using bioinformatics tools. Finally, the screening procedure improved from the first step was applied for screening Azurin-like genes, followed by the protein synthesis and NCI in vitro screening for anticancer activity. As a result, this study has successfully optimized the annealing temperatures to amplify DNAs for screening Azurin genes and applying to Azurin-like genes from human gut microbiota. The novelty of this study is the first of its kind to classify Azurin genes into five different genotypes at a global scale and confirm the potential anticancer activity of three Azurin-like synthetic proteins (Cnazu1, Dlazu11, and Ruazu12). The results contribute to the procedure development applied for screening anticancer proteins from human microbiome and a comprehensive understanding of their therapeutic response at a genetic level.

In the growing field of biomolecular electronics, blue-copper Azurin stands out as one of the most widely studied protein in single-molecule contacts. Interestingly, despite the paramount importance of the structure/dynamics of molecular contacts in their transport properties, these factors remain largely unexplored from the theoretical point of view in the context of single Azurin junctions. Here we address this issue using all-atom Molecular Dynamics (MD) of Pseudomonas Aeruginosa Azurin adsorbed to a Au(111) substrate. In particular, we focus on the structure and dynamics of the free/adsorbed protein and how these properties are altered upon single-point mutations. The results revealed that wild-type Azurin adsorbs on Au(111) along two well defined configurations: one tethered via cysteine groups and the other via the hydrophobic pocket surrounding the Cu 2 + . Surprisingly, our simulations revealed that single amino-acid mutations gave rise to a quenching of protein vibrations ultimately resulting in its overall stiffening. Given the role of amino-acid vibrations and reorientation in the dehydration process at the protein-water-substrate interface, we suggest that this might have an effect on the adsorption process of the mutant, giving rise to new adsorption configurations.

eMap is a web-based platform for identifying and visualizing electron or hole transfer pathways in proteins based on their crystal structures. The underlying model can be viewed as a coarse-grained version of the Pathways model, where each tunneling step between hopping sites represented by electron transfer active (ETA) moieties is described with one effective decay parameter that describes protein-mediated tunneling. ETA moieties include aromatic amino acid residue side chains and aromatic fragments of cofactors that are automatically detected, and, in addition, electron/hole residing sites that can be specified by the users. The software searches for the shortest paths connecting the user-specified electron/hole source to either all surface-exposed ETA residues or the user-specified target. The identified pathways are ranked according to their effective length. The pathways are visualized in 2D as a graph, in which each node represents an ETA site, and in 3D using available protein visualization tools. Here, we present the capability and user interface of eMap 1.0, which is available at https://emap.bu.edu .

Nickel-containing enzymes such as methyl coenzyme M reductase (MCR) and carbon monoxide dehydrogenase/acetyl coenzyme A synthase (CODH/ACS) play a critical role in global energy conversion reactions, with significant contributions to carbon-centered processes. These enzymes are implied to cycle through a series of nickel-based organometallic intermediates during catalysis, though identification of these intermediates remains challenging. In this work, we have developed and characterized a nickel-containing metalloprotein that models the methyl-bound organometallic intermediates proposed in the native enzymes. Using a nickel(I)-substituted azurin mutant, we demonstrate that alkyl binding occurs via nucleophilic addition of methyl iodide as a methyl donor. The paramagnetic Ni III -CH 3 species initially generated can be rapidly reduced to a high-spin Ni II -CH 3 species in the presence of exogenous reducing agent, following a reaction sequence analogous to that proposed for ACS. These two distinct bioorganometallic species have been characterized by optical, EPR, XAS, and MCD spectroscopy, and the overall mechanism describing methyl reactivity with nickel azurin has been quantitatively modeled using global kinetic simulations. A comparison between the nickel azurin protein system and existing ACS model compounds is presented. Ni III -CH 3 Az is only the second example of two-electron addition of methyl iodide to a Ni I center to give an isolable species and the first to be formed in a biologically relevant system. These results highlight the divergent reactivity of nickel across the two intermediates, with implications for likely reaction mechanisms and catalytically relevant states in the native ACS enzyme.

As one of the most prevalent malignancies, breast cancer still remains a significant risk for public health. Common therapeutic strategies include invasive surgery, chemotherapy and anti-herceptin antibodies. Adverse effects, drug resistance and low efficacy of current therapies necessitates the emergence of more effective platforms. Naturally released by the immune system, granzyme B activates multiple pro-apoptotic pathways by cleaving critical substrates. Bacterial cupredoxin, azurin, selectively targets cancer cells via a p53-dependent pathway. Fused by a linker, GrB-Azurin fusion protein was overexpressed in HEK293T cells, and purified by metal chromatography. SDS-PAGE, Western blotting and ELISA were performed to confirm successful expression, purification and analyze binding properties of the fusion protein. After treatment of various breast cancer cell lines with increasing concentrations of GrB-Azurin, quantitative real-time RT-PCR was used to measure relative expression of p21, Fas and DR5 pro-apoptotic genes. The results of DNA fragmentation and WST-1 cell viability assays indicated significant apoptosis induction in MDA-MB-231, MCF7 and SK-BR-3 cells, while insignificant cytotoxicity was detected on MCF 10A normal breast cells. Herein, we report the development of a novel biotherapeutic against breast cancer. Selective effectiveness of GrB-Azurin fusion protein on different breast cancer cells highlighted the potential of the designed construct as a candidate anti-cancer biodrug.

We have investigated photoinduced hole hopping in a Pseudomonas aeruginosa azurin mutant Re126WWCu I , where two adjacent tryptophan residues (W124 and W122) are inserted between the Cu I center and a Re photosensitizer coordinated to a H126 imidazole (Re = Re I (H126)(CO) 3 (dmp) + , dmp = 4,7-dimethyl-1,10-phenanthroline). Optical excitation of this mutant in aqueous media (≤40 μM) triggers 70 ns electron transport over 23 Å, yielding a long-lived (120 μs) Re I (H126)(CO) 3 (dmp •- )WWCu II product. The Re126FWCu I mutant (F124, W122) is not redox-active under these conditions. Upon increasing the concentration to 0.2-2 mM, {Re126WWCu I } 2 and {Re126FWCu I } 2 are formed with the dmp ligand of the Re photooxidant of one molecule in close contact (3.8 Å) with the W122' indole on the neighboring chain. In addition, {Re126WWCu I } 2 contains an interfacial tryptophan quadruplex of four indoles (3.3-3.7 Å apart). In both mutants, dimerization opens an intermolecular W122' → //*Re ET channel (// denotes the protein interface, *Re is the optically excited sensitizer). Excited-state relaxation and ET occur together in two steps (time constants of ∼600 ps and ∼8 ns) that lead to a charge-separated state containing a Re(H126)(CO) 3 (dmp •- )//(W122 •+ )' unit; then (Cu I )' is oxidized intramolecularly (60-90 ns) by (W122 •+ )', forming Re I (H126)(CO) 3 (dmp •- )WWCu I //(Cu II )'. The photocycle is closed by ∼1.6 μs Re I (H126)(CO) 3 (dmp •- ) → //(Cu II )' back ET that occurs over 12 Å, in contrast to the 23 Å, 120 μs step in Re126WWCu I . Importantly, dimerization makes Re126FWCu I photoreactive and, as in the case of {Re126WWCu I } 2 , channels the photoproduced "hole" to the molecule that was not initially photoexcited, thereby shortening the lifetime of Re I (H126)(CO) 3 (dmp •- )//Cu II . Although two adjacent W124 and W122 indoles dramatically enhance Cu I → *Re intramolecular multistep ET, the tryptophan quadruplex in {Re126WWCu I } 2 does not accelerate intermolecular electron transport; instead, it acts as a hole storage and crossover unit between inter- and intramolecular ET pathways. Irradiation of {Re126WWCu II } 2 or {Re126FWCu II } 2 also triggers intermolecular W122' → //*Re ET, and the Re(H126)(CO) 3 (dmp •- )//(W122 •+ )' charge-separated state decays to the ground state by ∼50 ns Re I (H126)(CO) 3 (dmp •- ) + → //(W122 •+ )' intermolecular charge recombination. Our findings shed light on the factors that control interfacial hole/electron hopping in protein complexes and on the role of aromatic amino acids in accelerating long-range electron transport.

In this study, azurin, a bacteriocin with anticancer property, was produced by food-grade Lactococcus lactis using the Nisin Controlled Gene Expression (NICE) System. In addition, the antibacterial and cytotoxic properties of recombinant azurin in the culture supernatant were also investigated. Azurin gene from Pseudomonas aeruginosa was cloned into the pNZ8149 vector and the resulting recombinant DNA was transformed into food grade L. lactis NZ3900. The expression of azurin protein was induced by the optimum concentration of nisin for 3 h. Inhibition zones for Escherichia coli and Bacillus cereus were observed at 5.0 and 10 mg/mL concentrations of lyophilized supernatants containing azurin, but no inhibition zone at azurin-free lyophilized supernatants. When HUVEC, HT29, HCT116, and MCF7 cell lines were treated with lyophilized culture supernatants with azurin or without azurin, cell viability decreased with increasing concentrations of the supernatant. Furthermore, the supernatants containing azurin showed more anti-proliferative effect than the azurin-free supernatants. This work provides a practicable method to produce recombinant azurin in the food grade L. lactis strain. As a result, the recombinant L. lactis strain, producing azurin, can be used in the investigation of food biopreservatives and in the development of a therapeutic probiotic.

Understanding the regulation mechanism and molecular determinants of the reduction potential of metalloprotein is a major challenge. An ab initio quantum mechanical/molecular mechanical (QM/MM) method combining the minimum free energy path (MFEP) and fractional number of electron (FNE) approaches has been developed in our group to simulate the redox processes of large systems. The FNE scheme provides an efficient unique description for the redox process, while the MFEP method provides improved conformational sampling on complex environments such as protein in the QM/MM calculations. The reduction potentials of wild-type and seven mutants of azurin, a type 1 copper metalloprotein, were simulated with the QM/MM-MFEP+FNE approach in this paper. A range of 350 mV for the variations of the reduction potentials of these azurin proteins was reproduced faithfully with relative errors around 20 mV. The correlation between structural interactions and reduction potentials observed in simulations provides in-depth insight into the regulation of reduction potentials, which potentially can also be very useful to the engineering of metalloprotein-based electrocatalysts in artificial photosynthesis. The excellent accuracy and efficiency of the QM/MM-MFEP+FNE approach demonstrate the potential for simulations of many electron transfer processes in condensed phases and biochemical systems.

N-heterocyclic carbene (NHC) ligands have had a major impact in homogeneous catalysis, however, their potential role in biological systems is essentially unexplored. We replaced a copper-coordinating histidine (His) in the active site of the redox enzyme azurin with exogenous dimethyl imidazolylidene. This NHC rapidly restores the type-1 Cu center, with spectroscopic properties (EPR, UV/Vis) that are identical to those from N-coordination of the His in the wild type. However, the introduction of the NHC markedly alters the redox potential of the metal, which is a key functionality of this blue copper protein. These results suggest that C-bonding for histidine is plausible and a potentially relevant bonding mode of redox-active metalloenzymes in their (transient) active states., (© 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Membrane lipid rafts are highly ordered microdomains and essential components of plasma membranes. In this work, we demonstrate that azurin uptake by cancer cells is, in part, mediated by caveolin-1 and GM-1, lipid rafts' markers. This recognition is mediated by a surface exposed hydrophobic core displayed by azurin since the substitution of a phenylalanine residue in position 114 facing the hydrophobic cavity by alanine impacts such interactions, debilitating the uptake of azurin by cancer cells. Treating of cancer cells with azurin leads to a sequence of events: alters the lipid raft exposure at plasma membranes, causes a decrease in the plasma membrane order as examined by Laurdan two-photon imaging and leads to a decrease in the levels of caveolin-1. Caveolae, a subset of lipid rafts characterized by the presence of caveolin-1, are gaining increasing recognition as mediators in tumor progression and resistance to standard therapies. We show that azurin inhibits growth of cancer cells expressing caveolin-1, and this inhibition is only partially observed with mutant azurin. Finally, the simultaneous administration of azurin with anticancer therapeutic drugs (paclitaxel and doxorubicin) results in an enhancement in their activity, contrary to the mutated protein.

Blue copper proteins, such as azurin, show dramatic changes in Cu 2+ /Cu + reduction potential upon mutation over the full physiological range. Hence, they have important functions in electron transfer and oxidation chemistry and have applications in industrial biotechnology. The details of what determines these reduction potential changes upon mutation are still unclear. Moreover, it has been difficult to model and predict the reduction potential of azurin mutants and currently no unique procedure or workflow pattern exists. Furthermore, high-level computational methods can be accurate but are too time consuming for practical use. In this work, a novel approach for calculating reduction potentials of azurin mutants is shown, based on a combination of continuum electrostatics, density functional theory and empirical hydrophobicity factors. Our method accurately reproduces experimental reduction potential changes of 30 mutants with respect to wildtype within experimental error and highlights the factors contributing to the reduction potential change. Finally, reduction potentials are predicted for a series of 124 new mutants that have not yet been investigated experimentally. Several mutants are identified that are located well over 10 Å from the copper center that change the reduction potential by more than 85 mV. The work shows that secondary coordination sphere mutations mostly lead to long-range electrostatic changes and hence can be modeled accurately with continuum electrostatics., (© 2017 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA.)

Bioelectronics moves toward designing nanoscale electronic platforms that allow in vivo determinations. Such devices require interfacing complex biomolecular moieties as the sensing units to an electronic platform for signal transduction. Inevitably, a systematic design goes through a bottom-up understanding of the structurally related electrical signatures of the biomolecular circuit, which will ultimately lead us to tailor its electrical properties. Toward this aim, we show here the first example of bioengineered charge transport in a single-protein electrical contact. The results reveal that a single point-site mutation at the docking hydrophobic patch of a Cu-azurin causes minor structural distortion of the protein blue Cu site and a dramatic change in the charge transport regime of the single-protein contact, which goes from the classical Cu-mediated two-step transport in this system to a direct coherent tunneling. Our extensive spectroscopic studies and molecular-dynamics simulations show that the proteins' folding structures are preserved in the single-protein junction. The DFT-computed frontier orbital of the relevant protein segments suggests that the Cu center participation in each protein variant accounts for the different observed charge transport behavior. This work is a direct evidence of charge transport control in a protein backbone through external mutagenesis and a unique nanoscale platform to study structurally related biological electron transfer.

Noncovalent, weak interactions in the second coordination sphere of the copper active site of Pseudoazurin (PAz) from Achromobacter cycloclastes were examined using a series of Met16X variants. In this study, the differences in protein stability due to the changes in the nature of the 16th amino acid (Met, Phe, Val, Ile) were investigated by electrospray ionization mass spectrometry (ESI-MS) and far-UV circular dichroism (CD) as a result of acid denaturation. The percentage of native states (folded holo forms) of Met16Phe variants was estimated to be 75% at pH 2.9 although the wild-type (WT), Met16Val and Met16Ile PAz, became completely unfolded. The high stability under acidic conditions is correlated with the result of the active site being stabilized by the aromatic substitution of the Met16 residue. The π-π interaction in the second coordination sphere makes a significant contribution to the stability of active site and the protein matrix., (© 2017 The Protein Society.)

Mononuclear cupredoxins contain a type 1 copper center with a trigonal or tetragonal geometry usually maintained by four ligands, a cystein, two histidines and a methionine. The recent discovery of new members of this family with unusual properties demonstrates, however, the versatility of this class of proteins. Changes in their ligand set lead to drastic variation in their metal site geometry and in the resulting spectroscopic and redox features. In our work, we report the identification of the copper ligands in the recently discovered cupredoxin AcoP. We show that even though AcoP possesses a classical copper ligand set, it has a highly perturbed copper center. In depth studies of mutant's properties suggest a high degree of constraint existing in the copper center of the wild type protein and even the addition of exogenous ligands does not lead to the reconstitution of the initial copper center. Not only the chemical nature of the axial ligand but also constraints brought by its covalent binding to the protein backbone might be critical to maintain a green copper site with high redox potential. This work illustrates the importance of experimentally dissecting the molecular diversity of cupredoxins to determine the molecular determinants responsible for their copper center geometry and redox potential., (Copyright © 2017 Elsevier B.V. All rights reserved.)

In the present study, we developed a biohybrid material composed of recombinant azurin and CdSe-ZnS quantum dot to perform as a resistive random access memory (ReRAM) device. Site specific amino acid sequences were introduced in azurin to bind with the surface of CdSe-ZnS nanoparticle allowing the formation of a hybrid and voltage-driven switching enabled to develop a resistive random access memory (ReRAM) device. The analytical measurements confirmed that the azurin and CdSe-ZnS nanoparticles were well conjugated and formed into a single hybrid. Further, reversible, bistable switching along with repeatable writing-reading-erasing processes on individual azurin/CdSe-ZnS hybrid at nanoscale was achieved on the hybrid device. The device was programmed tested for 50 cycles with an ON/OFF ratio and measured to be of three orders of magnitude. The developed device shown good stability and repeatability and operates at low voltages thus makes it promising candidate for future memory device applications., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Pseudoazurin from the denitrifying bacterium Alcaligenes faecalis (AfPAz) is a blue copper protein and functions as an electron donor to copper-containing nitrite reductase (CuNIR). Conventionally, AfPAz has been crystallized using highly concentrated ammonium sulfate as a precipitant. Here, a needle-like crystal of AfPAz grown in a solution containing a macromolecular precipitant, polyethylene glycol 8000 (PEG 8000), is reported. The crystal belonged to space group P6 1 , with unit-cell parameters a = b = 68.7, c = 94.2 Å. The structure has been determined and refined at 2.6 Å resolution. The asymmetric unit contained two AfPAz molecules contacting each other on negatively charged surfaces. The molecular packing of the crystal showed a right-handed double-helical arrangement of AfPAz molecules and hence of blue copper sites. This structure provides insight into the excluded-volume effect of PEG and the manner of assembly of AfPAz.

Type 1 copper (T1Cu) proteins are electron transfer (ET) proteins involved in many important biological processes. While the effects of changing primary and secondary coordination spheres in the T1Cu ET function have been extensively studied, few report has explored the effect of the overall protein structural perturbation on active site configuration or reduction potential of the protein, even though the protein scaffold has been proposed to play a critical role in enforcing the entatic or "rack-induced" state for ET functions. We herein report circular permutation of azurin by linking the N- and C-termini and creating new termini in the loops between 1 st and 2 nd β strands or between 3 rd and 4 th β strands. Characterization by electronic absorption, electron paramagnetic spectroscopies, as well as crystallography and cyclic voltammetry revealed that, while the overall structure and the primary coordination sphere of the circular permutated azurins remain the same as those of native azurin, their reduction potentials increased by 18 and 124 mV over that of WTAz. Such increases in reduction potentials can be attributed to subtle differences in the hydrogen-bonding network in secondary coordination sphere around the T1Cu center., (© 2016 The Protein Society.)

Heme peptides and their derivatives, also called microperoxidases (MPs), are employed as heme protein active site models, catalysts, and charge-transfer chromophores. In this work, two approaches to the biosynthesis of novel MPs are described. In one method, heme peptides are expressed as C-terminal tags to the protein azurin and the MP is liberated by proteolytic cleavage by an endopeptidase. In an alternative approach, heme peptides are expressed as N-terminal tags to the cysteine protease domain (CPD) of the Vibrio cholerae MARTX toxin. Once activated by inositol hexakisphosphate, CPD undergoes autocleavage at an N-terminal leucine residue to liberate the MP. Purification is aided by use of a histidine-immobilized Sepharose column that binds exposed heme [Asher, W. A., and Bren, K. L. (2010) Protein Sci. 19, 1830-1839]. These methods provide efficient and adaptable routes to the preparation of a wide range of biosynthetic heme peptides.

The role of a backbone carbonyl interaction with an engineered Cu A center in azurin was investigated by developing a method of synthesis and incorporation of a depsipeptide where one of the amide bonds in azurin is replaced by an ester bond using expressed protein ligation. Studies by electronic absorption and electron paramagnetic resonance spectroscopic techniques indicate that, while the substitution does not significantly alter the geometry of the site, it weakens the axial interaction to the Cu A center and strengthens the Cu-Cu bond, as evidenced by the blue shift of the near-IR absorption that has been assigned to the Cu-Cu ψ → ψ* transition. Interestingly, the changes in the electronic structure from the replacement did not result in a change in the reduction potential of the Cu A center, suggesting that the diamond core structure of Cu 2 S Cys2 is resistant to variations in axial interactions.

Nitrite reductase (NiR) activity was examined in a series of dicopper P.a. azurin variants in which a surface binding copper site was added through site-directed mutagenesis. Four variants were synthesized with copper binding motifs inspired by the catalytic type 2 copper binding sites found in the native noncoupled dinuclear copper enzymes nitrite reductase and peptidylglycine α-hydroxylating monooxygenase. The four azurin variants, denoted Az-NiR, Az-NiR3His, Az-PHM, and Az-PHM3His, maintained the azurin electron transfer copper center, with the second designed copper site located over 13 Å away and consisting of mutations Asn10His,Gln14Asp,Asn16His-azurin, Asn10His,Gln14His,Asn16His-azurin, Gln8Met,Gln14His,Asn16His-azurin, and Gln8His,Gln14His,Asn16His-azurin, respectively. UV-visible absorption spectroscopy, EPR spectroscopy, and electrochemistry of the sites demonstrate copper binding as well as interaction with small exogenous ligands. The nitrite reduction activity of the variants was determined, including the catalytic Michaelis-Menten parameters. The variants showed activity (0.34-0.59 min(-1)) that was slower than that of native NiRs but comparable to that of other model systems. There were small variations in activity of the four variants that correlated with the number of histidines in the added copper site. Catalysis was found to be reversible, with nitrite produced from NO. Reactions starting with reduced azurin variants demonstrated that electrons from both copper centers were used to reduce nitrite, although steady-state catalysis required the T2 copper center and did not require the T1 center. Finally, experiments separating rates of enzyme reduction from rates of reoxidation by nitrite demonstrated that the reaction with nitrite was rate limiting during catalysis.

In this work, we characterized the intermolecular electron transfer (ET) properties of a de novo designed metallopeptide using laser-flash photolysis. α3D-CH3 is three helix bundle peptide that was designed to contain a copper ET site that is found in the β-barrel fold of native cupredoxins. The ET activity of Cuα3D-CH3 was determined using five different photosensitizers. By exhibiting a complete depletion of the photo-oxidant and the successive formation of a Cu(II) species at 400 nm, the transient and generated spectra demonstrated an ET transfer reaction between the photo-oxidant and Cu(I)α3D-CH3. This observation illustrated our success in integrating an ET center within a de novo designed scaffold. From the kinetic traces at 400 nm, first-order and bimolecular rate constants of 10(5) s(-1) and 10(8) M(-1) s(-1) were derived. Moreover, a Marcus equation analysis on the rate versus driving force study produced a reorganization energy of 1.1 eV, demonstrating that the helical fold of α3D requires further structural optimization to efficiently perform ET. This article is part of a Special Issue entitled Biodesign for Bioenergetics--the design and engineering of electronic transfer cofactors, proteins and protein networks, edited by Ronald L. Koder and J.L. Ross Anderson., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Neisseria gonorrhoeae colonizes the genitourinary track, and in these environments, especially in the female host, the bacteria are subjected to low levels of oxygen, and reactive oxygen and nitrosyl species. Here, the biochemical characterization of N. gonorrhoeae Laz is presented, as well as, the solution structure of its soluble domain determined by NMR. N. gonorrhoeae Laz is a type 1 copper protein of the azurin-family based on its spectroscopic properties and structure, with a redox potential of 277±5 mV, at pH7.0, that behaves as a monomer in solution. The globular Laz soluble domain adopts the Greek-key motif, with the copper center located at one end of the β-barrel coordinated by Gly48, His49, Cys113, His118 and Met122, in a distorted trigonal geometry. The edge of the His118 imidazole ring is water exposed, in a surface that is proposed to be involved in the interaction with its redox partners. The heterologously expressed Laz was shown to be a competent electron donor to N. gonorrhoeae cytochrome c peroxidase. This is an evidence for its involvement in the mechanism of protection against hydrogen peroxide generated by neighboring lactobacilli in the host environment., (Copyright © 2015 Elsevier B.V. All rights reserved.)

The wide range of variability of the reduction potential (E(0)) of blue-copper proteins has been the subject of a large number of studies in the past several years. In particular, a series of azurin mutants have been recently rationally designed tuning E(0) over a very broad range (700 mV) without significantly altering the redox-active site [Marshall et al., Nature, 2009, 462, 113]. This clearly suggests that interactions outside the primary coordination sphere are relevant to determine E(0) in cupredoxins. However, the molecular determinants of the redox potential variability are still undisclosed. Here, by means of atomistic molecular dynamics simulations and hybrid quantum/classical calculations, the mechanisms that determine the E(0) shift of two azurin mutants with high potential shifts are unravelled. The reduction potentials of native azurin and of the mutants are calculated obtaining results in good agreement with the experiments. The analysis of the simulations reveals that only a small number of residues (including non-mutated ones) are relevant in determining the experimentally observed E(0) variation via site-specific, but diverse, mechanisms. These findings open the path to the rational design of new azurin mutants with different E(0).

Tryptophan and tyrosine can form radical intermediates that enable long-range, multistep electron transfer (ET) reactions in proteins. This report describes the mechanisms of formation and quenching of a neutral tryptophan radical in azurin, a blue-copper protein that contains native tyrosine (Y108 and Y72) and tryptophan (W48) residues. A long-lived neutral tryptophan radical W48• is formed upon UV-photoexcitation of a zinc(II)-substituted azurin mutant in the presence of an external electron acceptor. The quantum yield of W48• formation (Φ) depends upon the tyrosine residues in the protein. A tyrosine-deficient mutant, Zn(II)Az48W, exhibited a value of Φ = 0.080 with a Co(III) electron acceptor. A nearly identical quantum yield was observed when the electron acceptor was the analogous tyrosine-free, copper(II) mutant; this result for the Zn(II)Az48W:Cu(II)Az48W mixture suggests there is an interprotein ET path. A single tyrosine residue at one of the native positions reduced the quantum yield to 0.062 (Y108) or 0.067 (Y72). Wild-type azurin with two tyrosine residues exhibited a quantum yield of Φ = 0.045. These data indicate that tyrosine is able to quench the tryptophan radical in azurin.

The laz gene of Neisseria meningitidis is predicted to encode a lipid-modified azurin (Laz). Laz is very similar to azurin, a periplasmic protein, which belongs to the copper-containing proteins in the cupredoxin superfamily. In other bacteria, azurin is an electron donor to nitrite reductase, an important enzyme in the denitrifying process. It is not known whether Laz could function as an electron transfer protein in this important pathogen. Laz protein was heterologously expressed in Escherichia coli and purified. Electrospray mass spectrometry indicated that the Laz protein contains one copper ion. Laz was shown to be redox-active in the presence of its redox center copper ion. When oxidized, Laz exhibits an intense blue colour and absorbs visible light around 626 nm. The absorption is lost when exposed to diethyldithiocarbamate, a copper chelating agent. Polyclonal antibodies were raised against purified Laz for detecting expression of Laz under different growth conditions and to determine the orientation of Laz on the outer membrane. The expression of Laz under microaerobic and microaerobic denitrifying conditions was slightly higher than that under aerobic conditions. However, the expression of Laz was similar between the wild type strain and an fnr mutant, suggesting that Fumarate/Nitrate reduction regulator (FNR) does not regulate the expression of Laz despite the presence of a partial FNR box upstream of the laz gene. We propose that some Laz protein is exposed on the outer membrane surface of N. meningitidis as the αLaz antibodies can increase killing by complement in a capsule deficient N. meningitidis strain, in a dose-dependent fashion.

Cupredoxin-like proteins are mainly copper-binding proteins that conserve a typical rigid Greek-key arrangement consisting of an eight-stranded β-sandwich, even though they share as little as 10-15% sequence similarity. The electron transport function of the Cupredoxins is critical for respiration and photosynthesis, and the proteins have therapeutic potential. Despite their crucial biological functions, the identification of the distant Cupredoxin homologs has been a difficult task due to their low sequence identity. In this study, the overlapped conserved residue (OCR) fingerprint for the Cupredoxin superfamily, which consists of conserved residues in three aspects (i.e., the sequence, structure, and intramolecular interaction), was used to detect the novel Cupredoxin homologs in the NCBI non-redundant protein sequence database. The OCR fingerprint could identify 54 potential Cupredoxin sequences, which were validated by scanning them against the conserved Cupredoxin motif near the Cu-binding site. This study also attempted to model the 3D structures and to predict the functions of the identified potential Cupredoxins. This study suggests that the OCR-based approach can be used efficiently to detect novel homologous proteins with low sequence identity, such as Cupredoxins.

Mercury(II) metallation of Pseudomonas aeruginosa azurin has been characterized structurally and biochemically. The X-ray crystal structure at 1.5Å of mercury(II) metallated azurin confirms the coordination of mercury at the copper binding active site and a second surface site. These findings are further validated by NMR, Matrix-assisted laser desorption/ionization spectrometry (MALDI), and UV-visible spectroscopic methods indicating copper displacement from the wild-type protein. Bioinformatic analysis has identified homologous human protein domains computationally, and compared them to the structure of azurin, providing a model for human mercury interactions. Study of the mercury-azurin adduct, in combination with other known examples of protein-heavy metal interactions, could provide further insight into the chemical mechanisms of toxicological interactions, leading toward a global understanding of the biological speciation of toxic heavy metals., (Copyright © 2014. Published by Elsevier Inc.)

Much progress has been made in designing heme and dinuclear nonheme iron enzymes. In contrast, engineering mononuclear nonheme iron enzymes is lagging, even though these enzymes belong to a large class that catalyzes quite diverse reactions. Herein we report spectroscopic and X-ray crystallographic studies of Fe(II)-M121E azurin (Az), by replacing the axial Met121 and Cu(II) in wild-type azurin (wtAz) with Glu and Fe(II), respectively. In contrast to the redox inactive Fe(II)-wtAz, the Fe(II)-M121EAz mutant can be readily oxidized by Na2IrCl6, and interestingly, the protein exhibits superoxide scavenging activity. Mössbauer and EPR spectroscopies, along with X-ray structural comparisons, revealed similarities and differences between Fe(II)-M121EAz, Fe(II)-wtAz, and superoxide reductase (SOR) and allowed design of the second generation mutant, Fe(II)-M121EM44KAz, that exhibits increased superoxide scavenging activity by 2 orders of magnitude. This finding demonstrates the importance of noncovalent secondary coordination sphere interactions in fine-tuning enzymatic activity.