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Mycobacterium tuberculosis MhuD catalyzes the oxygenation of heme to mycobilin; experimental data presented here elucidates the novel hydroxylation reaction catalyzed by this enzyme. Analogues for the critical ferric-hydroperoxoheme (MhuD-heme-OOH) intermediate of this enzyme were characterized using UV/Vis absorption (Abs), circular dichroism (CD), and magnetic CD (MCD) spectroscopies. In order to extract electronic transition energies from these spectroscopic data, a novel global fitting model was developed for analysis of UV/Vis Abs, CD, and MCD data. A variant of MhuD was prepared, N7S, which weakens the affinity of heme-bound enzyme for a hydroperoxo analogue, azide, without significantly altering the protein secondary structure. Global fitting of spectroscopic data acquired in this study revealed that the second-sphere N7S substitution perturbs the electronic structure of two analogues for MhuD-heme-OOH: azide-inhibited MhuD (MhuD-heme-N 3 ) and cyanide-inhibited MhuD (MhuD-heme-CN). The ground state electronic structures of MhuD-heme-N 3 and MhuD-heme-CN were assessed using variable-temperature, variable-field MCD. Altogether, these data strongly suggest that there is a hydrogen bond between the Asn7 side-chain and the terminal oxygen of the hydroperoxo ligand in MhuD-heme-OOH. As discussed herein, this finding supports a novel hydroxylation reaction mechanism where the Asn7 side-chain guides a transient hydroxyl radical derived from homolysis of the OO bond in MhuD-heme-OOH to the β- or δ-meso carbon of the porphyrin ligand yielding β- or δ-meso-hydroxyheme, respectively., 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 Elsevier Inc. All rights reserved.)

Over the past decade, fluorophores that exhibit "mega" Stokes shifts, defined to be Stokes shifts of greater than 100 nm, have gained considerable attention due to their potential technological applications. A subset of these fluorophores have Stokes shifts of at least 10,000 cm -1 , for whom we suggest the moniker "giga" Stokes shift. The majority of "giga" Stokes shifts reported in the literature arise from the twisted intramolecular charge transfer mechanism, but this mechanism does not fit empirical characterization of triazolopyridinium (TOP). This observation inspired a density functional theory (DFT) and time-dependent DFT study of TOP, and several related fluorophores, to elucidate the novel photophysical origin for the "giga" Stokes shift of TOP. The resulting computational models revealed that photoexcitation of TOP yields a zwitterionic excited state that undergoes significant structural relaxation prior to emission. Most notably, TOP has two orthogonal moieties in the ground state that adopt a coplanar geometry in the excited state. According to Hückel's rule, both the heterocycle and phenyl moieties of TOP should be aromatic in an orthogonal ground state. However, according to Baird's rule, these individual moieties should be anti-aromatic in the excited state. By relaxing to a coplanar conformation in the excited state, TOP likely forms a single aromatic system consisting of both the heterocycle and phenyl moieties.

Non-canonical heme oxygenases are enzymes that degrade heme to non-biliverdin products within bacterial heme iron acquisition pathways. These enzymes all contain a conserved second-sphere Trp residue that is essential for enzymatic turnover. Here, UV/Vis absorption (Abs) and circular dichroism (CD) spectroscopies were employed to show that the W67F variant of IsdG perturbs the heme substrate conformation. In general, a dynamic equilibrium between "planar" and "ruffled" substrate conformations exists within non-canonical heme oxygenases, and that the second-sphere Trp favors population of the "ruffled" substrate conformation. 1 H nuclear magnetic resonance and magnetic CD spectroscopies were used to characterize the electronic structures of IsdG and IsdI variants with different substrate conformational distributions. These data revealed that the "ruffled" substrate conformation promotes partial porphyrin-to‑iron electron transfer, which makes the meso carbons of the porphyrin ring susceptible to radical attack. Finally, UV/Vis Abs spectroscopy was utilized to quantify the enzymatic rates, and electrospray ionization mass spectrometry was used to identify the product distributions, for variants of IsdG with altered substrate conformational distributions. In general, the rate of heme oxygenation by non-canonical heme oxygenases depends upon the population of the "ruffled" substrate conformation. Also, the production of staphylobilin or mycobilin by these enzymes is correlated with the population of the "ruffled" substrate conformation, since variants that favor population of the "planar" substrate conformation yield significant amounts of biliverdin. These data can be understood within the framework of a concerted rearrangement mechanism for the monooxygenation of heme to meso-hydroxyheme by non-canonical heme oxygenases., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Diruthenium paddlewheel complexes supported by electron-rich anilinopyridinate (Xap) ligands were synthesized in the course of the first in-depth structural and spectroscopic interrogation of monocationic [Ru 2 (Xap) 4 Cl] + species in the Ru 2 6+ oxidation state. Despite paramagnetism of the compounds, 1 H NMR spectroscopy proved highly informative for determining the isomerism of the Ru 2 5+ and Ru 2 6+ compounds. While most compounds are found to have the polar (4,0) geometry, with all four Xap ligands in the same orientation, some synthetic procedures resulted in a mixture of (4,0) and (3,1) isomers, most notably in the case of the parent compound Ru 2 (ap) 4 Cl. The isomerism of this compound has been overlooked in previous reports. Electrochemical studies demonstrate that oxidation potentials can be tuned by the installation of electron donating groups to the ligands, increasing accessibility of the Ru 2 6+ oxidation state. The resulting Ru 2 6+ monocations were found to have the expected (π*) 2 ground state, and an in-depth study of the electronic transitions by Vis/NIR absorption and MCD spectroscopies with the aid of TD-DFT allowed for the assignment of the electronic spectra. The empty δ* orbital is the major acceptor orbital for the most prominent electronic transitions. Both Ru 2 5+ and Ru 2 6+ compounds were studied by Ru K-edge X-ray absorption spectroscopy; however, the rising edge energy is insensitive to redox changes in the compounds due to the broad line shape observed for 4d transition metal K-edges. DFT calculations indicate the presence of ligand orbitals at the frontier level, suggesting that further oxidation beyond Ru 2 6+ will be ligand-centered rather than metal-centered.

Vitamin B 12 is one of the most complex cofactors known, and this chapter will discuss current understanding with regards to the cobalt insertion step of its syntheses. Two total syntheses of vitamin B 12 were reported in the 1970s, which remain two of the most exceptional achievements of natural product synthesis. In subsequent years, two distinct biosynthetic pathways were identified in aerobic and anaerobic organisms. For these biosynthetic pathways, selectivity for Co(II) over other divalent metal ions with similar ionic radii and coordination chemistry remains an open question with three competing hypotheses proposed: metal affinity, tetrapyrrole distortion, and product inhibition. A 20 step biosynthetic route to convert 5-aminolevulinic acid (ALA) to vitamin B 12 was elucidated in aerobic organisms in the 1990s, where cobalt is inserted relatively late in the pathway by the CobNST multi-protein complex. This chapter includes a mechanistic proposal for this reaction, but the majority of the proposal is based upon analogy to the ChlDHI magnesium chelatase complex as critical data for the cobalt chelatase is lacking. Later, in the 2010s, a distinct 21 step pathway from ALA to vitamin B 12 was reported in anaerobic organisms, where cobalt is inserted early in the pathway by the enzyme CbiK. A recent study strongly suggests that the cobalt affinity of CbiK is the origin of cobalt selectivity for CbiK, but several important mechanistic questions remain unanswered. In general, it is expected that significant insight into the cobalt insertion mechanisms of CobNST and CbiK could be derived from additional structural, spectroscopic, and computational data., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Ergothioneine (EGT) is the betaine of 2-thiohistidine (2-thioHis) and may be the last undiscovered vitamin. EGT cannot be incorporated into a peptide because the α-nitrogen is trimethylated, although this would be advantageous as an EGT-like moiety in a peptide would impart unique antioxidant and metal chelation properties. The amino acid 2-thioHis is an analogue of EGT and can be incorporated into a peptide, although there is only one reported occurrence of this in the literature. A likely reason is the harsh conditions reported for protection of the thione, with similarly harsh conditions used in order to achieve deprotection after synthesis. Here, we report a novel strategy for the incorporation of 2-thioHis into peptides in which we decided to leave the thione unprotected. This decision was based upon the reported low reactivity of EGT with 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB), a very electrophilic disulfide. This strategy was successful, and we report here the synthesis of 2-thioHis analogues of carnosine (βAH), GHK-tripeptide, and HGPLGPL. Each of these peptides contain a histidine (His) residue and possesses biological activity. Our results show that substitution of His with 2-thioHis imparts strong antioxidant, radical scavenging, and copper binding properties to the peptide. Notably, we found that the 2-thioHis analogue of GHK-tripeptide was able to completely quench the hydroxyl and ABTS radicals in our assays, and its antioxidant capacity was significantly greater than would be expected based on the antioxidant capacity of free 2-thioHis. Our work makes possible greater future use of 2-thioHis in peptides., (© 2021 European Peptide Society and John Wiley & Sons, Ltd.)

The noncanonical heme oxygenase MhuD from Mycobacterium tuberculosis binds a heme substrate that adopts a dynamic equilibrium between planar and out-of-plane ruffled conformations. MhuD degrades this substrate to an unusual mycobilin product via successive monooxygenation and dioxygenation reactions. This article establishes a causal relationship between heme substrate dynamics and MhuD-catalyzed heme degradation, resulting in a refined enzymatic mechanism. UV/vis absorption (Abs) and electrospray ionization mass spectrometry (ESI-MS) data demonstrated that a second-sphere substitution favoring the population of the ruffled heme conformation changed the rate-limiting step of the reaction, resulting in a measurable buildup of the monooxygenated meso-hydroxyheme intermediate. In addition, UV/vis Abs and ESI-MS data for a second-sphere variant that favored the planar substrate conformation showed that this change altered the enzymatic mechanism resulting in an α-biliverdin product. Single-turnover kinetic analyses for three MhuD variants revealed that the rate of heme monooxygenation depends upon the population of the ruffled substrate conformation. These kinetic analyses also revealed that the rate of meso-hydroxyheme dioxygenation by MhuD depends upon the population of the planar substrate conformation. Thus, the ruffled heme conformation supports rapid heme monooxygenation by MhuD, but further oxygenation to the mycobilin product is inhibited. In contrast, the planar substrate conformation exhibits altered heme monooxygenation regiospecificity followed by rapid oxygenation of meso-hydroxyheme. Altogether, these data yielded a refined enzymatic mechanism for MhuD where access to both substrate conformations is needed for rapid incorporation of three oxygen atoms into heme yielding mycobilin.

The nickel chelatase CfbA is the smallest member of the chelatase family, but the mechanism by which this enzyme inserts nickel into sirohydrochlorin is unknown. In order to gain mechanistic insight, metal binding, tetrapyrrole binding, and enzyme activity were characterized for a variety of substrates using several spectroscopic and computational approaches. Mass spectrometery and magnetic circular dichroism experiments revealed that CfbA binds an octahedral, high-spin metal substrate. UV/Vis absorption spectroscopy demonstrated that the enzyme binds a wide range of tetrapyrrole substrates and perturbs their electronic structures. Based upon activity assays, CfbA promotes insertion of cobalt and nickel into several tetrapyrroles, including cobalt insertion into protopophyrin IX. Finally, density functional theory models were developed which strongly suggest that observed spectral changes upon binding to the enzyme can be explained by tetrapyrrole ruffling, but not deprotonation or saddling. The observation of an octahedral, high-spin metal bound to CfbA leads to a generalization for all class II chelatases: these enzymes bind labile metal substrates and metal desolvation is not a rate-limiting step. The conclusion that CfbA ruffles its tetrapyrrole substrate reveals that the CfbA mechanism is different from that currently proposed for ferrochelatase, and identifies an intriguing correlation between metal substrate specificity and tetrapyrrole distortion mode in chelatases.

Staphylococcus aureus IsdG catalyzes a unique trioxygenation of heme to staphylobilin, and the data presented in this article elucidate the mechanism of the novel chemical transformation. More specifically, the roles of the second-sphere Asn and Trp residues in the monooxygenation of ferric-peroxoheme have been clarified via spectroscopic characterization of the ferric-azidoheme analogue. Analysis of UV/vis absorption data quantified the strength of the hydrogen bond that exists between the Asn7 side chain and the azide moiety of ferric-azidoheme. X-band electron paramagnetic resonance data were acquired and analyzed, which revealed that this hydrogen bond weakens the π-donor strength of the azide, resulting in perturbations of the Fe 3d based orbitals. Finally, nuclear magnetic resonance characterization of 13 C-enriched samples demonstrated that the Asn7···N 3 hydrogen bond triggers partial porphyrin to iron electron transfer, resulting in spin density delocalization onto the heme meso carbons. These spectroscopic experiments were complemented by combined quantum mechanics/molecular mechanics computational modeling, which strongly suggested that the electronic structure changes observed for the N7A variant arose from loss of the Asn7···N 3 hydrogen bond as opposed to a decrease in porphyrin ruffling. From these data a fascinating picture emerges where an Asn7···N 3 hydrogen bond is communicated through four bonds, resulting in meso carbons with partial cationic radical character that are poised for hydroxylation. This chemistry is not observed in other heme proteins because Asn7 and Trp67 must work in concert to trigger the requisite electronic structure change.

Two hydrazones featuring a unique excitation wavelength-dependent dual fluorescence emission have been developed. The mixing extent of the two emission bands can be modulated by tuning the excitation wavelength, resulting in multicolor and even white light emission from structurally simple hydrazones.

MhuD is a protein found in mycobacteria that can bind up to two heme molecules per protein monomer and catalyze the degradation of heme to mycobilin in vitro. Here the Kd1 for heme dissociation from heme-bound MhuD was determined to be 7.6 ± 0.8 nM and the Kd2 for heme dissocation from diheme-bound MhuD was determined to be 3.3 ± 1.1 μM. These data strongly suggest that MhuD is a competent heme oxygenase in vivo.

Idiopathic pulmonary fibrosis is characterized by excessive deposition of collagen in the lung, leading to chronically impaired gas exchange and death 1-3 . Oxidative stress is believed to be critical in this disease pathogenesis 4-6 , although the exact mechanisms remain enigmatic. Protein S-glutathionylation (PSSG) is a post-translational modification of proteins that can be reversed by glutaredoxin-1 (GLRX) 7 . It remains unknown whether GLRX and PSSG play a role in lung fibrosis. Here, we explored the impact of GLRX and PSSG status on the pathogenesis of pulmonary fibrosis, using lung tissues from subjects with idiopathic pulmonary fibrosis, transgenic mouse models and direct administration of recombinant Glrx to airways of mice with existing fibrosis. We demonstrate that GLRX enzymatic activity was strongly decreased in fibrotic lungs, in accordance with increases in PSSG. Mice lacking Glrx were far more susceptible to bleomycin- or adenovirus encoding active transforming growth factor beta-1 (AdTGFB1)-induced pulmonary fibrosis, whereas transgenic overexpression of Glrx in the lung epithelium attenuated fibrosis. We furthermore show that endogenous GLRX was inactivated through an oxidative mechanism and that direct administration of the Glrx protein into airways augmented Glrx activity and reversed increases in collagen in mice with TGFB1- or bleomycin-induced fibrosis, even when administered to fibrotic, aged animals. Collectively, these findings suggest the therapeutic potential of exogenous GLRX in treating lung fibrosis.

The micromolar equilibrium constants for heme dissociation from IsdG and IsdI reported in the literature call into question whether these enzymes are actually members of the iron-regulated surface determinant system of Staphylococcus aureus, which harvests heme iron from a host during infection. In order to address this question, the heme dissociation constants for IsdG and IsdI were reevaluated using three approaches. The heme dissociation equilibrium constants were measured using a UV/Vis absorption-detected assay analyzed with an assumption-free model, and using a newly developed fluorescence-detected assay. The heme dissociation rate constants were estimated using apomyoglobin competition assays. Analyses of the UV/Vis absorption data revealed a critical flaw in the previous measurements; heme is 99.9% protein-bound at the micromolar concentrations needed for UV/Vis absorption spectroscopy, which renders accurate equilibrium constant measurement nearly impossible. However, fluorescence can be measured for more dilute samples, and analyses of these data resulted in dissociation equilibrium constants of 1.4 ± 0.6 nM and 12.9 ± 1.3 nM for IsdG and IsdI, respectively. Analyses of the kinetic data obtained from apomyoglobin competition assays estimated heme dissociation rate constants of 0.022 ± 0.002 s -1 for IsdG and 0.092 ± 0.008 s -1 for IsdI. Based upon these data, and what is known regarding the post-translational regulation of IsdG and IsdI, it is proposed that only IsdG is a member of the heme iron acquisition pathway and IsdI regulates heme homeostasis. Furthermore, the nanomolar dissociation constants mean that heme is bound tightly by IsdG and indicates that competitive inhibition of this protein will be difficult. Instead, uncompetitive inhibition based upon a detailed understanding of enzyme mechanism is a more promising antibiotic development strategy.

Although there are some proposed explanations for aggregation-induced emission, a phenomenon with applications that range from biosensors to organic light-emitting diodes, current understanding of the quantum-mechanical origin of this photophysical behaviour is limited. To address this issue, we assessed the emission properties of a series of BF 2 -hydrazone-based dyes as a function of solvent viscosity. These molecules turned out to be highly efficient fluorescent molecular rotors. This property, in addition to them being aggregation-induced emission luminogens, enabled us to probe deeper into their emission mechanism. Time-dependent density functional theory calculations and experimental results showed that the emission is not from the S 1 state, as predicted from Kasha's rule, but from a higher energy (>S 1 ) state. Furthermore, we found that suppression of internal conversion to the dark S 1 state by restricting the rotor rotation enhances fluorescence, which leads to the proposal that suppression of Kasha's rule is the photophysical mechanism responsible for emission in both viscous solution and the solid state.

Correction for 'Dynamic ruffling distortion of the heme substrate in non-canonical heme oxygenase enzymes' by Amanda B. Graves et al., Dalton Trans., 2016, 45, 10058-10067.

Recent work by several groups has established that MhuD, IsdG, and IsdI are non-canonical heme oxygenases that induce significant out-of-plane ruffling distortions of their heme substrates enroute to mycobilin or staphylobilin formation. However, clear explanations for the observations of "nested" S = ½ VTVH MCD saturation magnetization curves at cryogenic temperatures, and exchange broadened (1)H NMR resonances at physiologically-relevant temperatures have remained elusive. Here, MCD and NMR data have been acquired for F23A and F23W MhuD-heme-CN, in addition to MCD data for IsdI-heme-CN, in order to complete assembly of a library of spectroscopic data for cyanide-inhibited ferric heme with a wide range of ruffling deformations. The spectroscopic data were used to evaluate a number of computational models for cyanide-inhibited ferric heme, which ultimately led to the development of an accurate NEVPT2/CASSCF model. The resulting model has a shallow, double-well potential along the porphyrin ruffling coordinate, which provides clear explanations for the unusual MCD and NMR data. The shallow, double-well potential also implies that MhuD-, IsdG-, and IsdI-bound heme is dynamic, and the functional implications of these dynamics are discussed.

For decades it has been known that an out-of-plane ruffling distortion of heme perturbs its UV-vis absorption (Abs) spectrum, but whether increased ruffling induces a red or blue shift of the Soret band has remained a topic of debate. This debate has been resolved by the spectroscopic and computational characterization of Mycobacterium tuberculosis MhuD presented here, an enzyme that converts heme, oxygen, and reducing equivalents to nonheme iron and mycobilin. W66F and W66A MhuD have been characterized using (1)H nuclear magnetic resonance, Abs, and magnetic circular dichroism spectroscopies, and the data have been used to develop an experimentally validated theoretical model of ruffled, ferric heme. The PBE density functional theory (DFT) model that has been developed accurately reproduces the observed spectral changes from wild type enzyme, and the underlying quantum mechanical origins of these ruffling-induced changes were revealed by analyzing the PBE DFT description of the electronic structure. Small amounts of heme ruffling have no influence on the energy of the Q-band and blue-shift the Soret band due to symmetry-allowed mixing of the Fe 3dxy and porphyrin a2u orbitals. Larger amounts of ruffling red-shift both the Q and Soret bands due to disruption of π-bonding within the porphyrin ring.

Staphylococcus aureus IsdG catalyzes the final step of staphylococcal iron acquisition from host hemoglobin, whereby host-derived heme is converted to iron and organic products. The Asn7 distal pocket residue is known to be critical for enzyme activity, but the influence of this residue on the substrate electronic structure was unknown prior to this work. Here, an optical spectroscopic and density functional theory characterization of azide- and cyanide-inhibited wild type and N7A IsdG is presented. Magnetic circular dichroism data demonstrate that Asn7 perturbs the electronic structure of azide-inhibited, but not cyanide-inhibited, IsdG. As the iron-ligating α-atom of azide, but not cyanide, can act as a hydrogen bond acceptor, these data indicate that the terminal amide of Asn7 is a hydrogen bond donor to the α-atom of a distal ligand to heme in IsdG. Circular dichroism characterization of azide- and cyanide-inhibited forms of WT and N7A IsdG strongly suggests that the Asn7···N3 hydrogen bond influences the orientation of a distal azide ligand with respect to the heme substrate. Specifically, density functional theory calculations suggest that Asn7···N3 hydrogen bond donation causes the azide ligand to rotate about an axis perpendicular to the porphyrin plane and weakens the π-donor strength of the azide ligand. This lowers the energies of the Fe 3d xz and 3d yz orbitals, mixes Fe 3d xy and porphyrin a 2u character into the singly-occupied molecular orbital, and results in spin delocalization onto the heme meso carbons. These discoveries have important implications for the mechanism of heme oxygenation catalyzed by IsdG.

Mycobacterium heme utilization degrader (MhuD) is a heme-degrading protein from Mycobacterium tuberculosis responsible for extracting the essential nutrient iron from host-derived heme. MhuD has been previously shown to produce unique organic products compared to those of canonical heme oxygenases (HOs) as well as those of the IsdG/I heme-degrading enzymes from Staphylococcus aureus. Here, we report the X-ray crystal structure of cyanide-inhibited MhuD (MhuD-heme-CN) as well as detailed (1)H nuclear magnetic resonance (NMR), UV/vis absorption, and magnetic circular dichroism (MCD) spectroscopic characterization of this species. There is no evidence for an ordered network of water molecules on the distal side of the heme substrate in the X-ray crystal structure, as was previously reported for canonical HOs. The degree of heme ruffling in the crystal structure of MhuD is greater than that observed for HO and less than that observed for IsdI. As a consequence, the Fe 3dxz-, 3dyz-, and 3dxy-based MOs are very close in energy, and the room-temperature (1)H NMR spectrum of MhuD-heme-CN is consistent with population of both a (2)Eg electronic state with a (dxy)(2)(dxz,dyz)(3) electron configuration, similar to the ground state of canonical HOs, and a (2)B2g state with a (dxz,dyz)(4)(dxy)(1) electron configuration, similar to the ground state of cyanide-inhibited IsdI. Variable temperature, variable field MCD saturation magnetization data establishes that MhuD-heme-CN has a (2)B2g electronic ground state with a low-lying (2)Eg excited state. Our crystallographic and spectroscopic data suggest that there are both structural and electronic contributions to the α-meso regioselectivity of MhuD-catalyzed heme cleavage. The structural distortion of the heme substrate observed in the X-ray crystal structure of MhuD-heme-CN is likely to favor cleavage at the α- and γ-meso carbons, whereas the spin density distribution may favor selective oxygenation of the α-meso carbon.

Mycobacterium tuberculosis is the causative agent of tuberculosis, which is becoming an increasingly global public health problem due to the rise of drug-resistant strains. While residing in the human host, M. tuberculosis needs to acquire iron for its survival. M. tuberculosis has two iron uptake mechanisms, one that utilizes non-heme iron and another that taps into the vast host heme-iron pool. To date, proteins known to be involved in mycobacterial heme uptake are Rv0203, MmpL3, and MmpL11. Whereas Rv0203 transports heme across the bacterial periplasm or scavenges heme from host heme proteins, MmpL3 and MmpL11 are thought to transport heme across the membrane. In this work, we characterize the heme-binding properties of the predicted extracellular soluble E1 domains of both MmpL3 and MmpL11 utilizing absorption, electron paramagnetic resonance, and magnetic circular dichroism spectroscopic methods. Furthermore, we demonstrate that Rv0203 transfers heme to both MmpL3-E1 and MmpL11-E1 domains at a rate faster than passive heme dissociation from Rv0203. This work elucidates a key step in the mycobacterial uptake of heme, and it may be useful in the development of anti-tuberculosis drugs targeting this pathway.

Two [1,2,3]triazolo[1,5-a]pyridinium salts have been prepared via a novel Cu(II)-mediated oxidative cyclization reaction. Both water soluble compounds exhibit broad-range fluorescence emission with huge Stokes shifts as well as color tunability.

Cytochrome c is a highly conserved protein, with 20 residues identical in all eukaryotic cytochromes c. Gly-41 is one of these invariant residues, and is the position of the only reported naturally occurring mutation in cytochrome c (human G41S). The basis, if any, for the conservation of Gly-41 is unknown. The mutation of Gly-41 to Ser enhances the apoptotic activity of cytochrome c without altering its role in mitochondrial electron transport. Here we have studied additional residue 41 variants and determined their effects on cytochrome c functions and conformation. A G41T mutation decreased the ability of cytochrome c to induce caspase activation and decreased the redox potential, whereas a G41A mutation had no impact on caspase induction but the redox potential increased. All residue 41 variants decreased the pK (a) of a structural transition of oxidized cytochrome c to the alkaline conformation, and this correlated with a destabilization of the interaction of Met-80 with the heme iron(III) at physiological pH. In reduced cytochrome c the G41T and G41S mutations had distinct effects on a network of hydrogen bonds involving Met-80, and in G41T the conformational mobility of two Ω-loops was altered. These results suggest the impact of residue 41 on the conformation of cytochrome c influences its ability to act in both of its physiological roles, electron transport and caspase activation.

The naturally occurring G41S mutation to human (Hs) cytochrome (cyt) c enhances apoptotic activity based upon previous in vitro and in vivo studies, but the molecular mechanism underlying this enhancement remains unknown. Here, X-ray crystallography, nuclear magnetic resonance (NMR) spectroscopy, and density functional theory (DFT) calculations have been used to identify the structural and electronic differences between wild-type (WT) and G41S Hs cyt c. S41 is part of the hydrogen bonding network for propionate 7 of heme pyrrole ring A in the X-ray structure of G41S Hs cyt c and, compared to WT, G41S Hs cyt c has increased spin density on pyrrole ring C and a faster electron self-exchange rate. DFT calculations illustrate an electronic mechanism where structural changes near ring A can result in electronic changes at ring C. Since ring C is part of the solvent-exposed protein surface, we propose that this heme electronic structure change may ultimately be responsible for the enhanced proapoptotic activity of G41S Hs cyt c.

Out-of-plane (OOP) deformations of the heme cofactor are found in numerous heme-containing proteins and the type of deformation tends to be conserved within functionally related classes of heme proteins. We demonstrate correlations between the heme ruffling OOP deformation and the (13)C and (1)H nuclear magnetic resonance (NMR) hyperfine shifts of heme aided by density functional theory (DFT) calculations. The degree of ruffling in the heme cofactor of Hydrogenobacter thermophilus cytochrome c(552) has been modified by a single amino acid mutation in the second coordination sphere of the cofactor. The (13)C and (1)H resonances of the cofactor have been assigned using one- and two-dimensional NMR spectroscopy aided by selective (13)C-enrichment of the heme. DFT has been used to predict the NMR hyperfine shifts and electron paramagnetic resonance (EPR) g-tensor at several points along the ruffling deformation coordinate. The DFT-predicted NMR and EPR parameters agree with the experimental observations, confirming that an accurate theoretical model of the electronic structure and its response to ruffling has been established. As the degree of ruffling increases, the heme methyl (1)H resonances move upfield while the heme methyl and meso (13)C resonances move downfield. These changes are a consequence of altered overlap of the Fe 3d and porphyrin pi orbitals, which destabilizes all three occupied Fe 3d-based molecular orbitals and decreases the positive and negative spin density on the beta-pyrrole and meso carbons, respectively. Consequently, the heme ruffling deformation decreases the electronic coupling of the cofactor with external redox partners and lowers the reduction potential of heme.

The one-electron-reduced form of vitamin B(12), cob(II)alamin (Co(2+)Cbl), is found in several essential human enzymes, including the cobalamin-dependent methionine synthase (MetH). In this work, experimentally validated electronic structure descriptions for two "base-off" Co(2+)Cbl species have been generated using a combined spectroscopic and computational approach, so as to obtain definitive clues as to how these and related enzymes catalyze the thermodynamically challenging reduction of Co(2+)Cbl to cob(I)alamin (Co(1+)Cbl). Specifically, electron paramagnetic resonance (EPR), electronic absorption (Abs), and magnetic circular dichroism (MCD) spectroscopic techniques have been employed as complementary tools to characterize the two distinct forms of base-off Co(2+)Cbl that can be trapped in the H759G variant of MetH, one containing a five-coordinate and the other containing a four-coordinate, square-planar Co(2+) center. Accurate spin Hamiltonian parameters for these low-spin Co(2+) centers have been determined by collecting EPR data using both X- and Q-band microwave frequencies, and Abs and MCD spectroscopic techniques have been employed to probe the corrin-centered pi --> pi* and Co-based d --> d excitations, respectively. By using these spectroscopic data to evaluate electronic structure calculations, we found that density functional theory provides a reasonable electronic structure description for the five-coordinate form of base-off Co(2+)Cbl. However, it was necessary to resort to a multireference ab initio treatment to generate a more realistic description of the electronic structure of the four-coordinate form. Consistent with this finding, our computational data indicate that, in the five-coordinate Co(2+)Cbl species, the unpaired spin density is primarily localized in the Co 3d(z(2))-based molecular orbital, as expected, whereas in the four-coordinate form, extensive Co 3d orbital mixing, configuration interaction, and spin-orbit coupling cause the unpaired electron to delocalize over several Co 3d orbitals. These results provide important clues to the mechanism of enzymatic Co(2+)Cbl --> Co(1+)Cbl reduction.

Because of their complex geometric and electronic structures, the active sites and cofactors of bioorganometallic enzymes, which are characterized by their metal-carbon bonds, pose a major challenge for computational chemists. However, recent progress in computer technology and theoretical chemistry, along with insights gained from mechanistic, spectroscopic, and X-ray crystallographic studies, have established an excellent foundation for the successful completion of computational studies aimed at elucidating the electronic structures and catalytic cycles of these species. This chapter briefly reviews the most popular computational approaches employed in theoretical studies of bioorganometallic species and summarizes important information obtained from computational studies of (i) the enzymatic formation and cleavage of the Co-C bond of coenzyme B(12); (ii) the catalytic cycle of methyl-coenzyme M reductase and its nickel-containing cofactor F(430); (iii) the polynuclear active-site clusters of the bifunctional enzyme carbon monoxide dehydrogenase/acetyl-coenzyme A synthase; and (iv) the magnetic properties of the active-site cluster of Fe-only hydrogenases.

The cobalamin-dependent methionine synthase (MetH) from Escherichia coli is a modular enzyme that catalyzes a methyl group transfer from methyltetrahydrofolate to homocysteine via a methylcob(III)alamin (MeCbl) intermediate, generating tetrahydrofolate and methionine (Met). Once every approximately 2000 turnovers, the cobalamin cofactor is converted to the inactive cob(II)alamin (Co(2+)Cbl) form, from which MeCbl has to be recovered for MetH to re-enter the catalytic cycle. A particularly puzzling aspect of this reactivation process is that it requires the reduction of the Co(2+)Cbl species to cob(I)alamin (Co(1+)Cbl) by flavodoxin, a reaction that would appear to be endergonic on the basis of the corresponding reduction potentials. To explore how MetH may overcome this apparent thermodynamic challenge, we have prepared the I690C/G743C variant of a C-terminal fragment of MetH (MetH(CT)) to lock the enzyme into the activation conformation without perturbing any of the residues in the vicinity of the active site. A detailed spectroscopic characterization of this species and the I690C/G743C/Y1139F MetH(CT) triple mutant reveals that the strategy employed by MetH to activate Co(2+)Cbl for Co(2+) --> Co(1+) reduction likely involves (i) an axial ligand switch to generate a five-coordinate species with an axially coordinated water molecule and (ii) a significant lengthening, or perhaps complete rupture, of the Co-OH(2) bond of the cofactor, thereby causing a large stabilization of the Co 3d(z(2))-based "redox-active" molecular orbital. The lengthening of the Co-OH(2) bond is mediated by the Y1139 active-site residue and becomes much more dramatic when the S-adenosylmethionine substrate is present in the enzyme active site. This substrate requirement provides MetH a means to suppress deleterious side reactions involving the transiently formed Co(1+)Cbl "supernucleophile".

Cobalamin-dependent methionine synthase (MetH) of Escherichia coli is a 136 kDa, modular enzyme that undergoes large conformational changes as it uses a cobalamin cofactor as a donor or acceptor in three separate methyl transfer reactions. At different points during the reaction cycle, the coordination to the cobalt of the cobalamin changes; most notably, the imidazole side chain of His759 that coordinates to the cobalamin in the "His-on" state can dissociate to produce a "His-off" state. Here, two distinct species of the cob(II)alamin-bound His759Gly variant have been identified and separated. Limited proteolysis with trypsin was employed to demonstrate that the two species differ in protein conformation. Magnetic circular dichroism and electron paramagnetic resonance spectroscopies were used to show that the two species also differ with respect to the axial coordination to the central cobalt ion of the cobalamin cofactor. One form appears to be in a conformation poised for reductive methylation with adenosylmethionine; this form was readily reduced to cob(I)alamin and subsequently methylated [albeit yielding a unique, five-coordinate methylcob(III)alamin species]. Our spectroscopic data revealed that this form contains a five-coordinate cob(II)alamin species, with a water molecule as an axial ligand to the cobalt. The other form appears to be in a catalytic conformation and could not be reduced to cob(I)alamin under any of the conditions tested, which precluded conversion to the methylcob(III)alamin state. This form was found to possess an effectively four-coordinate cob(II)alamin species that has neither water nor histidine coordinated to the cobalt center. The formation of this four-coordinate cob(II)alamin "dead-end" species in the His759Gly variant illustrates the importance of the His759 residue in governing the equilibria between the different conformations of MetH.

The 4-coordinate, low-spin cob(I)alamin (Co1+Cbl) species, which can be obtained by heterolytic cleavage of the Co-C bond in methylcobalamin or the two-electron reduction of vitamin B12, is one of the most powerful nucleophiles known to date. The supernucleophilicity of Co1+Cbl has been harnessed by a number of cobalamin-dependent enzymes, such as the B12-dependent methionine synthase, and by enzymes involved in the biosynthesis of B12, including the human adenosyltransferase. The nontoxic nature of the Co1+Cbl supernucleophile also makes it an attractive target for the in situ bioremediation of halogenated waste. To gain insight into the geometric, electronic, and vibrational properties of this highly reactive species, electronic absorption, circular dichroism (CD), magnetic CD, and resonance Raman (rR) spectroscopies have been employed in conjunction with density functional theory (DFT), time-dependent DFT, and combined quantum mechanics/molecular mechanics computations. Collectively, our results indicate that the supernucleophilicity of Co1+Cbl can be attributed to the large destabilization of the Co 3dz2-based HOMO and its favorable orientation with respect to the corrin macrocycle, which minimizes steric repulsion during nucleophilic attack. An intense feature in the CD spectrum and a prominent peak in the rR spectra of Co1+Cbl have been identified that may serve as excellent probes of the nucleophilic character, and thus the reactivity, of Co1+Cbl in altered environments, including enzyme active sites. The implications of our results with respect to the enzymatic formation and reactivity of Co1+Cbl are discussed, and spectroscopic trends along the series from Co3+Cbls to Co2+Cbl and Co1+Cbl are explored.

The GAUSSIAN 2, GAUSSIAN 3, complete basis set-QB3, and complete basis set-APNO methods have been used to calculate DeltaH( composite function) and DeltaG( composite function) values for ionic clusters of hydronium and hydroxide ions complexed with water. Results for the clusters H3O+(H2O)n and OH-(H2O)n, where n=1-4 are reported in this paper, and compared against experimental values contained in the National Institutes of Standards and Technology (NIST) database. Agreement with experiment is excellent for the three ab initio methods for formation of these clusters. The high accuracy of these methods makes them reliable for calculating energetics for the formation of ionic clusters containing water. In addition this allows them to serve as a valuable check on the accuracy of experimental data reported in the NIST database, and makes them useful tools for addressing unresolved issues in atmospheric chemistry., (2005 American Institute of Physics.)

The CBS-QB3 method was used to calculate the gas-phase free energy difference between 20 phenols and their respective anions, and the CPCM continuum solvation method was applied to calculate the free energy differences of solvation for the phenols and their anions. The CPCM solvation calculations were performed on both gas-phase and solvent-phase optimized structures. Absolute pK(a) calculations with solvated phase optimized structures for the CPCM calculations yielded standard deviations and root-mean-square errors of less than 0.4 pK(a) unit. This study is the most accurate absolute determination of the pK(a) values of phenols, and is among the most accurate of any such calculations for any group of compounds. The ability to make accurate predictions of pK(a) values using a coherent, well-defined approach, without external approximations or fitting to experimental data, is of general importance to the chemical community. The solvated phase optimized structures of the anions are absolutely critical to obtain this level of accuracy, and yield a more realistic charge separation between the negatively charged oxygen and the ring system of the phenoxide anions.

Complete Basis Set and Gaussian-n methods were combined with CPCM continuum solvation methods to calculate pK(a) values for six carboxylic acids. An experimental value of -264.61 kcal/mol for the free energy of solvation of H(+), DeltaG(s)(H(+)), was combined with a value for G(gas)(H(+)) of -6.28 kcal/mol to calculate pK(a) values with Cycle 1. The Complete Basis Set gas-phase methods used to calculate gas-phase free energies are very accurate, with mean unsigned errors of 0.3 kcal/mol and standard deviations of 0.4 kcal/mol. The CPCM solvation calculations used to calculate condensed-phase free energies are slightly less accurate than the gas-phase models, and the best method has a mean unsigned error and standard deviation of 0.4 and 0.5 kcal/mol, respectively. The use of Cycle 1 and the Complete Basis Set models combined with the CPCM solvation methods yielded pK(a) values accurate to less than half a pK(a) unit.