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In this study, we constructed a metal-binding site close to the heme cofactor in myoglobin (Mb) by covalently attaching a nonnative metal-binding ligand of bipyridine to Cys46 through the F46C mutation in the heme distal site. The X-ray structure of the designed enzyme, termed F46C-mBpy Mb, was solved in the Cu(II)-bound form, which revealed the formation of a heterodinuclear center of Cu-His-H 2 O-heme. Cu(II)-F46C-mBpy Mb exhibits not only nitrite reductase reactivity but also cascade reaction activity involving both hydrolysis and oxidation. Furthermore, F46C-mBpy Mb displays Mn-peroxidase activity by the oxidation of Mn 2+ to Mn 3+ using H 2 O 2 as an oxidant. This study shows that the construction of a nonnative metal-binding site close to the heme cofactor is a convenient approach to creating an artificial metalloenzyme with a heterodinuclear center that confers multiple functions.

Globins, such as myoglobin (Mb) and neuroglobin (Ngb), are ideal protein scaffolds for the design of functional metalloenzymes. To date, numerous approaches have been developed for enzyme design. This review presents a summary of the progress made in the design of functional metalloenzymes based on Mb and Ngb, with a focus on the exploitation of covalent interactions, including coordination bonds and covalent modifications. These include the construction of a metal-binding site, the incorporation of a non-native metal cofactor, the formation of Cys/Tyr-heme covalent links, and the design of disulfide bonds, as well as other Cys-covalent modifications. As exemplified by recent studies from our group and others, the designed metalloenzymes have potential applications in biocatalysis and bioconversions. Furthermore, we discuss the current trends in the design of functional metalloenzymes and highlight the importance of covalent interactions in the design of functional metalloenzymes., 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 © 2024 Elsevier Inc. All rights reserved.)

Using myoglobin (Mb) as a model protein, we herein developed a facial approach to modifying the heme active site. A cavity was first generated in the heme distal site by F46 C mutation, and the thiol group of Cys46 was then used for covalently linked to exogenous ligands, 1H-1,2,4-triazole-3-thiol and 1-(4-hydroxyphenyl)-1H-pyrrole-2,5-dione. The engineered proteins, termed F46C-triazole Mb and F46C-phenol Mb, respectively, were characterized by X-ray crystallography, spectroscopic and stopped-flow kinetic studies. The results showed that both the heme coordination state and the protein function such as H 2 O 2 activation and peroxidase activity could be efficiently regulated, which suggests that this approach might be generally applied to the design of functional heme proteins., (© 2023 Wiley-VCH GmbH.)

Significant efforts have been made in the design of artificial metalloenzymes. Myoglobin (Mb), an O 2 carrier, has been engineered to exhibit different functions. Herein, we applied a series of engineered Mb mutants with peroxidase activity for biosynthesis of clofazimine (CFZ), a potential drug with a broad-spectrum antiviral activity, by integration with chemical synthesis. Two of those mutants, F43Y Mb and F43Y/T67R Mb, have been shown to efficiently catalyze the oxidative coupling of 2- N -(4-chlorophenyl) benzene-1,2-diamine ( N -4-CPBDA) in the presence of H 2 O 2 , with 97% yields. The overall catalytic efficiency ( k cat / K m ) is 46-fold and 82-fold higher than that of WT Mb, respectively. By further combination of this reaction with chemical synthesis, the production of CFZ was accomplished with an isolated yield of 72%. These results showed that engineered Mbs containing the Tyr-heme cross-link (F43Y Mb and F43Y/T67R Mb) exhibit enhanced activity in the oxidative coupling reaction. This study also indicates that the combination of biocatalysis and chemical synthesis avoids the need for the separation of intermediate products, which offers a convenient approach for the total synthesis of the biological compound CFZ.

The design of functional metalloenzymes is attractive for the biosynthesis of biologically important compounds, such as phenoxazinones and phenazines catalyzed by native phenoxazinone synthase (PHS). To design functional heme enzymes, we used myoglobin (Mb) as a model protein and introduced an artificial CXXC motif into the heme distal pocket by F46C and L49C mutations, which forms a de novo disulfide bond, as confirmed by the X-ray crystal structure. We further introduced a catalytic Tyr43 into the heme distal pocket and found that the F43Y/F46C/L49C Mb triple mutant and the previously designed F43Y/F46S Mb exhibit PHS-like activity (80-98% yields in 5-15 min), with the catalytic efficiency exceeding those of natural metalloenzymes, including o -aminophenol oxidase, laccase, and dye-decolorizing peroxidase. Moreover, we showed that the oxidative coupling product of 1,6-disulfonic-2,7-diaminophenazine is a potential pH indicator, with the orange-magenta color change at pH 4-5 (p K a = 4.40). Therefore, this study indicates that functional heme enzymes can be rationally designed by structural modifications of Mb, exhibiting the functionality of the native PHS for green biosynthesis.

Tetracyclines are one class of widely used antibiotics. Meanwhile, due to abuse and improper disposal, they are often detected in wastewater, which causes a series of environmental problems and poses a threat to human health and safety. As an efficient and environmentally friendly method, enzymatic catalysis has attracted much attention. In previous studies, we have designed an efficient peroxidase (F43Y/P88W/F138W Mb, termed YWW Mb) based on the protein scaffold of myoglobin (Mb), an O 2 carrier, by modifying the heme active center and introducing two Trp residues. In this study, we further applied it to degrade the tetracycline antibiotics. Both UV-Vis and HPLC studies showed that the triple mutant YWW Mb was able to catalyze the degradation of tetracycline, oxytetracycline, doxycycline, and chlortetracycline effectively, with a degradation rate of ~100%, ~98%, ~94%, and ~90%, respectively, within 5 min by using H 2 O 2 as an oxidant. These activities are much higher than those of wild-type Mb and other heme enzymes such as manganese peroxidase. As further analyzed by UPLC-ESI-MS, we identified multiple degradation products and thus proposed possible degradation mechanisms. In addition, the toxicity of the products was analyzed by using in vitro antibacterial experiments of E. coli . Therefore, this study indicates that the engineered heme enzyme has potential applications for environmental remediation by degradation of tetracycline antibiotics.

Heme proteins perform a variety of biological functions and also play significant roles in the field of bio-catalysis. The β-lactamase activity of heme proteins has rarely been reported. Herein, we found, for the first time, that myoglobin (Mb), an O 2 carrier, also exhibits novel β-lactamase activity by catalyzing the hydrolysis of ampicillin. The catalytic proficiency (( k cat / K M )/ k uncat ) was determined to be 6.25 × 10 10 , which is much higher than the proficiency reported for designed metalloenzymes, although it is lower than that of natural β-lactamases. Moreover, we found that this activity could be regulated by an engineered disulfide bond, such as Cys46-Cys61 in F46C/L61C Mb or by the addition of imidazole to directly coordinate to the heme center. These results indicate that the heme active site is responsible for the β-lactamase activity of Mb. Therefore, the study suggests the potential of heme proteins acting as β-lactamases, which broadens the diversity of their catalytic functions.

Heme proteins play vital roles in regulating the reactive oxygen/nitrogen species (ROS/RNS) levels in cells. In this study, we overexpressed human wild-type (WT) myoglobin (Mb) and its double mutant, F43H/H64A Mb with enhanced nitrite reductase (NIR) activity, in the typical representative triple-negative breast cancer cell, MDA-MB-231 cells. The results showed that the overexpression of F43H/H64A Mb increased the level of nitric oxide (NO) and the degree of oxidative stress, and then activated Akt/MAPK mediated apoptotic cascade, whereas WT Mb showed the opposite effect. This study indicates that Mb plays an important role in maintaining the balance of the cellular redox system and could thus be a valuable target for cancer therapy., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Heme proteins have recently emerged as promising artificial metalloenzymes for catalyzing diverse reactions. In this report, L29E Mb, a single mutant of myoglobin (Mb), was reconstituted by replacing the heme with a sodium copper cholorophyllin (CuCP) to form a new green artificial enzyme (named CuCP-L29E Mb). The reconstituted protein CuCP-L29E Mb was found to exhibit hydrolytic DNA cleavage activity, which was not depending on O 2 . In addition, Mg 2+ ion could effectively promote the DNA cleavage activity of CuCP-L29E Mb. Wild-type (WT) Mb reconstituted with CuCP (named CuCP-WT Mb) did not show DNA cleavage activity under the same conditions. This study suggests that both Mg 2+ and the ligand Glu29 are critical for the nuclease activity and the artificial nuclease of Mg 2+ -CuCP-L29E Mb may have potential applications in the future., (Copyright © 2022. Published by Elsevier Inc.)

An artificial disulfide bond (Cys46-Cys61) was designed in the heme distal site of myoglobin, which regulates the conformation of the heme distal His64 and the protein reactivity, as confirmed by X-ray crystallography, EPR, and kinetic UV-vis studies. This study shows the successful design of a disulfide bond with suitable positions in globins, conferring a structure and function like those of the native human neuroglobin.

Globins are heme proteins such as hemoglobin (Hb), myoglobin (Mb) and neuroglobin (Ngb), playing important roles in biological system. In addition to normal functions, zebrafish Ngb was able to penetrate cell membranes, whereas less was known for other globin members. In this study, to improve the cell-membrane-penetrating activity of globins, we used sperm whale Mb as a model protein and constructed a quadruple mutant of G5K/Q8K/A19K/V21K Mb (termed 4K Mb), by introduction of four positive charges on the protein surface, which was designed according to the amino acid alignment with that of zebrafish Ngb. Spectroscopic and crystallographic studies showed that the four positively charged Lys residues did not affect the protein structure. Cell-membrane-penetrating essay further showed that 4K Mb exhibited enhanced activity compared to that of native Mb. This study provides valuable information for the effect of distribution of charged residues on the protein structure and the cell-membrane-penetrating activity of globins. Therefore, it will guide the design of protein-based biomaterials for biological applications., (Copyright © 2022 Elsevier Inc. All rights reserved.)

The treatment of environmental pollutants such as synthetic dyes and lignin has received much attention, especially for biotechnological treatments using both native and artificial metalloenzymes. In this study, we designed and engineered an efficient peroxidase using the O 2 carrier myoglobin (Mb) as a protein scaffold by four mutations (F43Y/T67R/P88W/F138W), which combines the key structural features of natural peroxidases such as the presence of a conserved His-Arg pair and Tyr/Trp residues close to the heme active center. Kinetic studies revealed that the quadruple mutant exhibits considerably enhanced peroxidase activity, with the catalytic efficiency ( k cat / K m ) comparable to that of the most efficient natural enzyme, horseradish peroxidase (HRP). Moreover, the designed enzyme can effectively decolorize a variety of synthetic organic dyes and catalyze the bioconversion of lignin, such as Kraft lignin and a model compound, guaiacylglycerol-β-guaiacyl ether (GGE). As analyzed by HPLC and ESI-MS, we identified several bioconversion products of GGE, as produced via bond cleavage followed by dimerization or trimerization, which illustrates the mechanism for lignin bioconversion. This study indicates that the designed enzyme could be exploited for the decolorization of textile wastewater contaminated with various dyes, as well as for the bioconversion of lignin to produce more value-added products.

Protein glycation is an important protein post-translational modification and is one of the main pathogenesis of diabetic angiopathy. Other than glycated hemoglobin, the protein glycation of other globins such as myoglobin (Mb) is less studied. The protein glycation of human Mb with ribose has not been reported, and the glycation sites in the Mb remain unknown. This article reports that d-ribose undergoes rapid protein glycation of human myoglobin (HMb) at lysine residues (K34, K87, K56, and K147) on the protein surface, as identified by ultra-high performance liquid chromatography-mass spectrometry (UHPLC-MS) and electrospray ionization tandem mass spectrometry (ESI-MS/MS). Moreover, glycation by d-ribose at these sites slightly decreased the rate of the met heme (Fe III ) in reaction with H 2 O 2 to form a ferryl heme (Fe IV =O). This study provides valuable insight into the protein glycation by d-ribose and provides a foundation for studying the structure and function of glycated heme proteins.

Inspired by the structural features of native peroxidases, an artificial peroxidase was rationally designed using F43Y myoglobin with a Tyr-heme cross-link by further introduction of key residues, including both a distal Arg and a Trp close to the heme group, which exhibits an enhanced peroxidase activity similar to the most efficient native horseradish peroxidase. This study provides a simple approach for design of artificial heme enzymes by the combination of catalytic elements of native enzymes with the post-translational modifications of heme proteins.

The X-ray crystal structure of F43Y/T67R myoglobin revealed unique Tyr-heme double cross-links between Tyr43 and the heme 4-vinyl group, which represents a novel post-translational modification of heme proteins. Moreover, with the feature of a distal His-Arg pair, the designed artificial enzyme exhibited a peroxidase activity comparable to that of native peroxidases, such as the most efficient horseradish peroxidase.

The structure and function of heme proteins are regulated by diverse post-translational modifications including heme-protein cross-links, with the underlying mechanisms not well understood. In this study, we introduced a Cys (K42C) close to the heme 4-vinyl group in sperm whale myoglobin (Mb) and solved its X-ray crystal structure. Interestingly, we found that K42C Mb can partially form a Cys-heme cross-link (termed K42C Mb-X) under dithiothreitol-induced reductive conditions in presence of O 2 , as suggested by guanidine hydrochloride-induced unfolding and heme extraction studies. Mass spectrometry (MS) studies, together with trypsin digestion studies, further indicated that a thioether bond is formed between Cys42 and the heme 4-vinyl group with an additional mass of 16 Da, likely due to hydroxylation of the α‑carbon. We then proposed a plausible mechanism for the formation of the novel Cys-heme cross-link based on MS, kinetic UV-vis and electron paramagnetic resonance (EPR) studies. Moreover, the Cys-heme cross-link was shown to fine-tune the protein reactivity toward activation of H 2 O 2 . This study provides valuable insights into the post-translational modification of heme proteins, and also suggests that the Cys-heme cross-link can be induced to form in vitro, making it useful for design of new heme proteins with a non-dissociable heme and improved functions., (Copyright © 2018 Elsevier Inc. All rights reserved.)

A de novo designed intramolecular disulfide bond in myoglobin, resembling that in cytoglobin without structural evidence, was confirmed by an X-ray structure for the first time and was demonstrated to regulate both the structure and function of this protein, which fulfills the design of an artificial dehaloperoxidase, with an activity exceeding that of a native enzyme.

The rational design of metalloenzymes provides advantages not only for illustrating the structure and function relationship of native enzymes, but also for creating functional artificial enzymes comparable to native enzymes. Dye-decolorizing peroxidases (DyPs) are a new family of heme peroxidases and have received much attention recently. Inspired by the structural features of native DyPs with multiple Tyr and Trp residues, we herein aimed to design functional artificial DyPs using myoglobin (Mb), an O 2 carrier as a protein scaffold, by further introduction of Tyr/Trp into the secondary sphere of the heme center in the F43Y Mb mutant. The latter has been shown to possess a novel Tyr-heme cross-link and exhibit enhanced peroxidase activity, which provides an ideal platform to design a series of derivatives, including F43Y/F46Y Mb, F43Y/I107Y Mb, F43Y/F138 W Mb and F43Y/I107Y/F138 W Mb. Our design revealed that the Tyr-heme cross-link was well-retained in the mutants except for F43Y/F46Y Mb, as confirmed by X-ray crystal structure analysis. More importantly, stopped-flow kinetic studies showed that these derivatives exhibit enhanced dye-decolorizing peroxidase activities compared to that of wild-type (WT) Mb. This is particularly the case for the double mutant F43Y/F138 W Mb, exhibiting an overall catalytic efficiency (k cat /K m ) of 110 670 M -1 s -1 , which is ∼144-fold and ∼20-fold that of WT Mb and F43Y Mb, respectively, and is ∼4.3-fold that of native DyP from Vibrio cholerae. Stopped-flow, electron paramagnetic resonance (EPR) and isothermal titration calorimetry (ITC) studies further provided insights into the activation of H 2 O 2 and the binding of a substrate, reactive blue 19 (RB19), to the double mutant. This study provides valuable information for elucidating the structure and dye-decolorizing function relationship of peroxidases, and also clues for the design of other functional artificial heme enzymes.

A hydrogen-bond (H-bond) network, specifically a Tyr-associated H-bond network, plays key roles in regulating the structure and function of proteins, as exemplified by abundant heme proteins in nature. To explore an approach for fine-tuning the structure and function of artificial heme proteins, we herein used myoglobin (Mb) as a model protein and introduced a Tyr residue in the secondary sphere of the heme active site at two different positions (107 and 138). We performed X-ray crystallography, UV-Vis spectroscopy, stopped-flow kinetics, and electron paramagnetic resonance (EPR) studies for the two single mutants, I107Y Mb and F138Y Mb, and compared to that of wild-type Mb under the same conditions. The results showed that both Tyr107 and Tyr138 form a distinct H-bond network involving water molecules and neighboring residues, which fine-tunes ligand binding to the heme iron and enhances the protein stability, respectively. Moreover, the Tyr107-associated H-bond network was shown to fine-tune both H2O2 binding and activation. With two cases demonstrated for Mb, this study suggests that the Tyr-associated H-bond network has distinct roles in regulating the protein structure, properties and functions, depending on its location in the protein scaffold. Therefore, it is possible to design a Tyr-associated H-bond network in general to create other artificial heme proteins with improved properties and functions.

Heme proteins perform diverse functions in living systems, of which nitrite reductase (NIR) activity receives much attention recently. In this study, to better understand the structural elements responsible for the NIR activity, we used myoglobin (Mb) as a model heme protein and redesigned the heme active center, by introducing one or two distal histidines, and by creating a channel to the heme center with removal of the native distal His64 gate (His to Ala mutation). UV-Vis kinetic studies, combined with EPR studies, showed that a single distal histidine with a suitable position to the heme iron, i.e., His43, is crucial for nitrite (NO2(-)) to nitric oxide (NO) reduction. Moreover, creation of a water channel to the heme center significantly enhanced the NIR activity compared to the corresponding mutant without the channel. In addition, X-ray crystallographic studies of F43H/H64A Mb and its complexes with NO2(-) or NO revealed a unique hydrogen-bonding network in the heme active center, as well as unique substrate and product binding models, providing valuable structural information for the enhanced NIR activity. These findings enriched our understanding of the structure and NIR activity relationship of heme proteins. The approach of creating a channel in this study is also useful for rational design of other functional heme proteins., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Disulfide bond plays crucial roles in stabilization of protein structure and in fine-tuning protein functions. To explore an approach for rational heme protein design, we herein rationally introduced a pair of cysteines (F46C/M55C) into the scaffold of myoglobin (Mb), mimicking those in native neuroglobin. Molecular modeling suggested that it is possible for Cys46 and Cys55 to form an intramolecular disulfide bond, which was confirmed experimentally by ESI-MS analysis, DTNB reaction and CD spectrum. Moreover, it was shown that the spontaneously formed disulfide bond of Cys46-Cys55 fine-tunes not only the heme active site structure, but also the protein functions. The substitution of Phe46 with Ser46 in F46S Mb destabilizes the protein while facilitates H2O2 activation. Remarkably, the formation of an intramolecular disulfide bond of Cys46-Cys55 in F46C/M55C Mb improves the protein stability and regulates the heme site to be more favorable for substrate binding, resulting in enhanced peroxidase activity. This study provides valuable information of structure-function relationship for heme proteins regulated by an intramolecular disulfide bond, and also suggests that construction of such a covalent bond is useful for design of functional heme proteins., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Heme proteins perform diverse biological functions, of which myoglobin (Mb) is a representative protein. In this study, the O2 carrier Mb was shown to cleave double stranded DNA upon aerobic dithiothreitol-induced reduction, which is fine-tuned by an additional distal histidine, His29 or His43, engineered in the heme active center. Spectroscopic (UV-vis and EPR) and inhibition studies suggested that free radicals including singlet oxygen and hydroxyl radical are responsible for efficient DNA cleavage via an oxidative cleavage mechanism. On the other hand, L29E Mb, with a distinct heme active center involving three water molecules in the met form, was found to exhibit an excellent DNA cleavage activity that was not depending on O2. Inhibition and ligation studies demonstrated for the first time that L29E Mb cleaves double stranded DNA into both the nicked circular and linear forms via a hydrolytic cleavage mechanism, which resembles native endonucleases. This study provides valuable insights into the distinct mechanisms for DNA cleavage by heme proteins, and lays down a base for creating artificial DNA endonucleases by rational design of heme proteins. Moreover, this study suggests that the diverse functions of heme proteins can be fine-tuned by rational design of the heme active center with a hydrogen-bonding network., (Copyright © 2016 Elsevier Inc. All rights reserved.)

A heme-protein cross-link is a key post-translational modification (PTM) of heme proteins. Meanwhile, the structural and functional consequences of heme-protein cross-links are not fully understood, due to limited studies on a direct comparison of the same protein with and without the cross-link. A Tyr-heme cross-link with a C-O bond is a newly discovered PTM of heme proteins, and is spontaneously formed in F43Y myoglobin (Mb) between the Tyr hydroxyl group and the heme 4-vinyl group in vivo. In this study, we found that with an additional distal His29 introduced in the heme pocket, the double mutant L29H/F43Y Mb can form two distinct forms under different protein purification conditions, with and without a novel Tyr-heme cross-link. By solving the X-ray structures of both forms of L29H/F43Y Mb and performing spectroscopic studies, we made a direct structural and functional comparison in the same protein scaffold. It revealed that the Tyr-heme cross-link regulates the heme distal hydrogen-bonding network, and fine-tunes not only the spectroscopic and ligand binding properties, but also the protein reactivity. Moreover, the formation of the Tyr-heme cross-link in the double mutant L29H/F43Y Mb was investigated in vitro. This study addressed the key issue of how Tyr-heme cross-link fine-tunes the structure and functions of the heme protein, and provided a plausible mechanism for the formation of the newly discovered Tyr-heme cross-link.

Protein design is a useful method to create novel artificial proteins. A rational approach to design a heterodimeric protein using domain swapping for horse myoglobin (Mb) was developed. As confirmed by X-ray crystallographic analysis, a heterodimeric Mb with two different active sites was produced efficiently from two surface mutants of Mb, in which the charges of two amino acids involved in the dimer salt bridges were reversed in each mutant individually, with the active site of one mutant modified. This study shows that the method of constructing heterodimeric Mb with domain swapping is useful for designing artificial multiheme proteins., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Heme post-translational modification plays a key role in tuning the structure and function of heme proteins. We herein report a novel tyrosine-heme covalent C−O bond in an artificially produced sperm whale myoglobin (Mb) mutant, F43Y Mb, which formed spontaneously in vivo between the Tyr43 hydroxy group and the heme 4-vinyl group. This highlights the diverse chemistry of heme post-translational modifications, and lays groundwork for further investigation of the structural and functional diversity of covalently-bound heme proteins., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Protein nitration is an important post-translational modification regulating protein structure and function, especially for heme proteins. Myoglobin (Mb) is an ideal protein model for investigating the structure and function relationship of heme proteins. With limited structural information available for nitrated heme proteins from experiments, we herein performed a molecular dynamics study of human Mb with successive nitration of Tyr103, Tyr146, Trp7 and Trp14. We made a detailed comparison of protein motions, intramolecular contacts and internal cavities of nitrated Mbs with that of native Mb. It showed that although nitration of both Tyr103 and Tyr146 slightly alters the local conformation of heme active site, further nitration of both Trp7 and Trp14 shifts helix A apart from the rest of protein, which results in altered internal cavities and forms a water channel, representing an initial stage of Mb unfolding. The computational study provides an insight into the nitration of heme proteins at an atomic level, which is valuable for understanding the structure and function relationship of heme proteins in non-native states by nitration., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

The interaction of blood glucose with heme proteins plays a key role in inducing diabetes, a serious disease threatening human health. In this study, we investigated the non-covalent interaction between glucose and myoglobin (Mb), both theoretically and experimentally, using molecular dynamics (MD) simulation combined with spectroscopic studies. It revealed that glucoses can occupy the side pocket of Mb, and bind closely to one of the xenon cavities in Mb, by hydrogen bonding interactions with two propionate groups of heme as well as surrounding amino acids. These interactions alter the conformation of the heme active site slightly and lead to an enhanced peroxidase activity of Mb, as determined by kinetic studies. This study provides general information for glucose-heme proteins interactions, and also for blood glucose-protein interactions for patients with diabetes.

Myoglobin (Mb) is an ideal scaffold protein for rational protein design mimicking native enzymes. We recently designed a nitrite reductase (NiR) based on sperm whale Mb by introducing an additional distal histidine (Leu29 to His29 mutation) and generating a distal tyrosine (Phe43 to Tyr43 mutation) in the heme pocket, namely L29H/F43Y Mb, to mimic the active site of cytochrome cd (1) NiR from Ps. aeruginosa that contains two distal histidines and one distal tyrosine. The molecular modeling and dynamics simulation study herein revealed that L29H/F43Y Mb has the necessary structural features of native cytochrome cd (1) NiR and can provide comparable interactions with nitrite as in native NiRs, which provides rationality for the protein design and guides the protein engineering. Additionally, the present study provides an insight into the relatively low NiR activity of Mb in biological systems.

Sperm whale myoglobin (swMb) is a well-studied heme protein, both experimentally and theoretically. Comparatively, little attention has been paid to another member of Mb family, Aplysia limacina myoglobin (apMb). swMb and apMb have the same overall structure and perform the same biological function, i.e., O(2) carrier, while using a distinct heme active site. To provide insights into the structure-function relationship for these two Mbs, we herein made a comparison in terms of their dynamics properties using molecular dynamics simulation. We analyzed the overall structure and protein motions, as well as the intramolecular contacts, namely salt-bridges and hydrogen bonds, especially the interactions between the heme propionate groups and the polypeptide chain. The internal cavities in apMb were also compared to the well-known xenon and other cavities in swMb. Based on current simulations, we propose a unique "arginine gate" for apMb, which has a similar function to the histidine gate observed for swMb in previous studies. This study provides valuable information for understanding the homology of heme proteins, and also aids in rational design of structural and functional heme proteins by alternating the heme active site.

Denitrifying NO reductases are evolutionarily related to the superfamily of heme--copper terminal oxidases. These transmembrane protein complexes utilize a heme-nonheme diiron center to reduce two NO molecules to N(2)O. To understand this reaction, the diiron site has been modeled using sperm whale myoglobin as a scaffold and mutating distal residues Leu-29 and Phe-43 to histidines and Val-68 to a glutamic acid to create a nonheme Fe(B) site. The impact of incorporation of metal ions at this engineered site on the reaction of the ferrous heme with one NO was examined by UV-vis absorption, EPR, resonance Raman, and FTIR spectroscopies. UV--vis absorption and resonance Raman spectra demonstrate that the first NO molecule binds to the ferrous heme, but while the apoproteins and Cu(I)- or Zn(II)-loaded proteins show characteristic EPR signatures of S = 1/2 six-coordinate heme {FeNO}(7) species that can be observed at liquid nitrogen temperature, the Fe(II)-loaded proteins are EPR silent at ≥30 K. Vibrational modes from the heme [Fe-N-O] unit are identified in the RR and FTIR spectra using (15)NO and (15)N(18)O. The apo and Cu(I)-bound proteins exhibit ν(FeNO) and ν(NO) that are only marginally distinct from those reported for native myoglobin. However, binding of Fe(II) at the Fe(B) site shifts the heme ν(FeNO) by 17 cm(-1) and the ν(NO) by -50 cm(-1) to 1549 cm(-1). This low ν(NO) is without precedent for a six-coordinate heme {FeNO}(7) species and suggests that the NO group adopts a strong nitroxyl character stabilized by electrostatic interaction with the nearby nonheme Fe(II). Detection of a similarly low ν(NO) in the Zn(II)-loaded protein supports this interpretation.

Rational design of functional enzymes is a powerful strategy to gain deep insights into more complex native enzymes, such as nitric oxide reductase (NOR). Recently, we engineered a functional model of NOR by creating a two His and one Glu (2-His-1-Glu) non-heme iron center in sperm whale myoglobin (swMb L29E, F43H, H64, called Fe(B)Mb(-His)). It was found that Fe(B) Mb(-His) adopts a low-spin state with bis-His coordination in the absence of metal ions binding to the designed metal center. However, no structural information was available for the variant in this special spin state. We herein performed molecular modeling of Fe(B)Mb(-His) and compared with the X-ray structure of its copper bound derivative, Cu(II)-CN(-)-Fe(B) Mb(-His), resolved recently at a high resolution (1.65 Å) (PDB entry 3MN0). The simulated structure shows that mutation of Leu to Glu at position 29 in the hydrophobic heme pocket alters the folding behavior of Mb. The hydrogen bond between Glu29 and His64 further plays a role in stabilizing the bis-His (His64/His93) coordination structure. This study offers an excellent example of using molecular modeling to gain insights in rational design of both structural and functional proteins., (Copyright © 2010 Wiley-Liss, Inc.)

A conserved 2-His-1-Glu metal center, as found in natural nonheme iron-containing enzymes, was engineered into sperm whale myoglobin by replacing Leu29 and Phe43 with Glu and His, respectively (swMb L29E, F43H, H64, called Fe(B)Mb(-His)). A high resolution (1.65 A) crystal structure of Cu(II)-CN(-)-Fe(B)Mb(-His) was determined, demonstrating that the unique 2-His-1-Glu metal center was successfully created within swMb. The Fe(B)Mb(-His) can bind Cu, Fe, or Zn ions, with both Cu(I)-Fe(B)Mb(-His) and Fe(II)-Fe(B)Mb(-His) exhibiting nitric oxide reductase (NOR) activities. Cu dependent NOR activity was significantly higher than that of Fe in the same metal binding site. EPR studies showed that the reduction of NO to N(2)O catalyzed by these two enzymes resulted in different intermediates; a five-coordinate heme-NO species was observed for Cu(I)-Fe(B)Mb(-His) due to the cleavage of the proximal heme Fe-His bond, while Fe(II)-Fe(B)Mb(-His) remained six-coordinate. Therefore, both the metal ligand, Glu29, and the metal itself, Cu or Fe, play crucial roles in NOR activity. This study presents a novel protein model of NOR and provides insights into a newly discovered member of the NOR family, gNOR.

A structural and functional model of bacterial nitric oxide reductase (NOR) has been designed by introducing two glutamates (Glu) and three histidines (His) in sperm whale myoglobin. X-ray structural data indicate that the three His and one Glu (V68E) residues bind iron, mimicking the putative Fe(B) site in NOR, while the second Glu (I107E) interacts with a water molecule and forms a hydrogen bonding network in the designed protein. Unlike the first Glu (V68E), which lowered the heme reduction potential by approximately 110 mV, the second Glu has little effect on the heme potential, suggesting that the negatively charged Glu has a different role in redox tuning. More importantly, introducing the second Glu resulted in a approximately 100% increase in NOR activity, suggesting the importance of a hydrogen bonding network in facilitating proton delivery during NOR reactivity. In addition, EPR and X-ray structural studies indicate that the designed protein binds iron, copper, or zinc in the Fe(B) site, each with different effects on the structures and NOR activities, suggesting that both redox activity and an intermediate five-coordinate heme-NO species are important for high NOR activity. The designed protein offers an excellent model for NOR and demonstrates the power of using designed proteins as a simpler and more well-defined system to address important chemical and biological issues.

Inspired by the structural features of native peroxidases, an artificial peroxidase was rationally designed using F43Y myoglobin with a Tyr-heme cross-link by further introduction of key residues, including both a distal Arg and a Trp close to the heme group, which exhibits an enhanced peroxidase activity similar to the most efficient native horseradish peroxidase. This study provides a simple approach for design of artificial heme enzymes by the combination of catalytic elements of native enzymes with the post-translational modifications of heme proteins. [ABSTRACT FROM AUTHOR]

Emerging pollutants in the aquatic environment from pharmaceutical and personal care products (PPCPs) attract much attention due to their environmental risk and toxicological properties, posing a threat to human health and safety. In this study, we fulfilled the direct and efficient degradation of PPCPs (i.e. diclofenac sodium, 2-mercaptobenzothiazole, paracetamol, and furosemide) using engineered H 2 O 2 -dependent heme enzymes based on myoglobin (Mb) with high peroxidase activity. Especially, the triple mutant F43Y/P88W/F138W Mb could efficiently degrade diclofenac sodium, 2-mercaptobenzothiazole, paracetamol, and furosemide with a degradation rate of ∼98.5%, ∼99%, ∼98%, and ∼91%, respectively. These activities are much higher than other reported native enzymes, such as laccase and horseradial peroxidase. As further analyzed by ESI-MS, we identified multiple degradation products of those four contaminants and thus proposed possible degradation mechanisms. This study indicates that enzyme-based degradation of emerging pollutants is a promising environmental remediation approach. [Display omitted] • Rationally designed and constructed heme enzymes exhibit high peroxidase activity. • The H 2 O 2 -dependent heme enzymes could efficiently degrade emerging PPCPs. • The interaction between heme enzyme and substrate was studied by molecular docking. • The mechanism of pollutant degradation catalyzed by the engineered heme enzymes was proposed. [ABSTRACT FROM AUTHOR]

The rational design of metalloenzymes provides advantages not only for illustrating the structure and function relationship of native enzymes, but also for creating functional artificial enzymes comparable to native enzymes. Dye-decolorizing peroxidases (DyPs) are a new family of heme peroxidases and have received much attention recently. Inspired by the structural features of native DyPs with multiple Tyr and Trp residues, we herein aimed to design functional artificial DyPs using myoglobin (Mb), an O2 carrier as a protein scaffold, by further introduction of Tyr/Trp into the secondary sphere of the heme center in the F43Y Mb mutant. The latter has been shown to possess a novel Tyr–heme cross-link and exhibit enhanced peroxidase activity, which provides an ideal platform to design a series of derivatives, including F43Y/F46Y Mb, F43Y/I107Y Mb, F43Y/F138 W Mb and F43Y/I107Y/F138 W Mb. Our design revealed that the Tyr–heme cross-link was well-retained in the mutants except for F43Y/F46Y Mb, as confirmed by X-ray crystal structure analysis. More importantly, stopped-flow kinetic studies showed that these derivatives exhibit enhanced dye-decolorizing peroxidase activities compared to that of wild-type (WT) Mb. This is particularly the case for the double mutant F43Y/F138 W Mb, exhibiting an overall catalytic efficiency (kcat/Km) of 110 670 M−1 s−1, which is ∼144-fold and ∼20-fold that of WT Mb and F43Y Mb, respectively, and is ∼4.3-fold that of native DyP from Vibrio cholerae. Stopped-flow, electron paramagnetic resonance (EPR) and isothermal titration calorimetry (ITC) studies further provided insights into the activation of H2O2 and the binding of a substrate, reactive blue 19 (RB19), to the double mutant. This study provides valuable information for elucidating the structure and dye-decolorizing function relationship of peroxidases, and also clues for the design of other functional artificial heme enzymes. [ABSTRACT FROM AUTHOR]

A heme–protein cross-link is a key post-translational modification (PTM) of heme proteins. Meanwhile, the structural and functional consequences of heme-protein cross-links are not fully understood, due to limited studies on a direct comparison of the same protein with and without the cross-link. A Tyr–heme cross-link with a C–O bond is a newly discovered PTM of heme proteins, and is spontaneously formed in F43Y myoglobin (Mb) between the Tyr hydroxyl group and the heme 4-vinyl group in vivo. In this study, we found that with an additional distal His29 introduced in the heme pocket, the double mutant L29H/F43Y Mb can form two distinct forms under different protein purification conditions, with and without a novel Tyr–heme cross-link. By solving the X-ray structures of both forms of L29H/F43Y Mb and performing spectroscopic studies, we made a direct structural and functional comparison in the same protein scaffold. It revealed that the Tyr–heme cross-link regulates the heme distal hydrogen-bonding network, and fine-tunes not only the spectroscopic and ligand binding properties, but also the protein reactivity. Moreover, the formation of the Tyr–heme cross-link in the double mutant L29H/F43Y Mb was investigated in vitro. This study addressed the key issue of how Tyr–heme cross-link fine-tunes the structure and functions of the heme protein, and provided a plausible mechanism for the formation of the newly discovered Tyr–heme cross-link. [ABSTRACT FROM AUTHOR]

Protein design is a useful method to create novel artificial proteins. A rational approach to design a heterodimeric protein using domain swapping for horse myoglobin (Mb) was developed. As confirmed by X-ray crystallographic analysis, a heterodimeric Mb with two different active sites was produced efficiently from two surface mutants of Mb, in which the charges of two amino acids involved in the dimer salt bridges were reversed in each mutant individually, with the active site of one mutant modified. This study shows that the method of constructing heterodimeric Mb with domain swapping is useful for designing artificial multiheme proteins. [ABSTRACT FROM AUTHOR]

Abstract: Heme proteins perform various biological functions ranging from electron transfer, oxygen binding and transport, catalysis, to signaling. Although adopting proper native states is very important for these functions, progresses in representative heme proteins, including cytochrome c (cyt c), cytochrome b 5 (cyt b 5), myoglobin (Mb), neuroglobin (Ngb), cytochrome P450 (CYP) and heme-based sensor proteins such as CO sensor CooA, showed that various native functions, or new functions evolved, are also closely associated with non-native states. The structure and function relationship of heme proteins in non-native states is thus as important as that in native states for elucidating the precise roles of heme proteins in biological systems. [Copyright &y& Elsevier]

Abstract: Rationally designed biosynthetic models provide fantastic advantages for addressing important issues in chemistry and biology. Previously, two distal histidines were introduced in the heme pocket of myoglobin (Mb) at positions 29 and 43. The resultant His29 and His43, together with the native His64, formed a metal-binding site in the designed L29H/F43H Mb mutant, which closely mimics the heterobinuclear center of native cytochrome c oxidase (CcO). Instead of studying the oxidase activity, we herein investigated the cross-reactivity, i.e., the peroxidase activity of this mutant, as well as the effect of metal ions binding to the designed metal-binding site on protein reactivity. It was found that the introduced two distal histidines with no bound metal ion serve to improve the peroxidase activity of L29H/F43H Mb compared with that of WT Mb containing a single distal histidine, whereas the binding of metal ions such as Cu(II) and Zn(II) to L29H/F43H Mb inhibits the catalytic activity to a different extent. These findings provide valuable insights into the peroxidase activity observed for the native CcO, as well as the role of metal ion in fine-tuning the protein cross-reactivity. [Copyright &y& Elsevier]

The treatment of environmental pollutants such as synthetic dyes and lignin has received much attention, especially for biotechnological treatments using both native and artificial metalloenzymes. In this study, we designed and engineered an efficient peroxidase using the O2 carrier myoglobin (Mb) as a protein scaffold by four mutations (F43Y/T67R/P88W/F138W), which combines the key structural features of natural peroxidases such as the presence of a conserved His-Arg pair and Tyr/Trp residues close to the heme active center. Kinetic studies revealed that the quadruple mutant exhibits considerably enhanced peroxidase activity, with the catalytic efficiency (kcat/Km) comparable to that of the most efficient natural enzyme, horseradish peroxidase (HRP). Moreover, the designed enzyme can effectively decolorize a variety of synthetic organic dyes and catalyze the bioconversion of lignin, such as Kraft lignin and a model compound, guaiacylglycerol-β-guaiacyl ether (GGE). As analyzed by HPLC and ESI-MS, we identified several bioconversion products of GGE, as produced via bond cleavage followed by dimerization or trimerization, which illustrates the mechanism for lignin bioconversion. This study indicates that the designed enzyme could be exploited for the decolorization of textile wastewater contaminated with various dyes, as well as for the bioconversion of lignin to produce more value-added products. [ABSTRACT FROM AUTHOR]

• Biodegradation of environmental pollutants has been favored for decades. • Significant progress has been made in the biodegradation of aromatic pollutants by metalloenzymes. • Both native and artificial metalloenzymes have been applied for biodegradation. • The progress sheds light on the structure-function relationship of metalloenzymes. • The progress provides clues for applications of metalloenzymes in environmental protection. Environmental security is closely related to public health. Aromatic pollutants constitute a class of hazardous environmental chemicals, such as halophenols and polycyclic aromatic hydrocarbons. Biodegradation of environmental pollutants has been favored for decades. As reviewed herein, significant progress has been made in biodegradation of aromatic pollutants by native metalloenzymes, including heme enzymes (peroxidases and cytochrome P450s), non-heme iron-containing enzymes (Rieske dioxygenases), and copper-containing enzymes (catechol oxidase, tyrosinase, and laccase), and artificial metalloenzymes, including engineered myoglobins, designed manganese peroxidase and de novo designed metalloenzymes, such as those in helical bundles and peptide assemblies, as well as synthetic structural and functional models of natural enzymes. These advances shed light on the structure–function relationship of metalloenzymes, providing clues for potential applications in environmental protection. Future directions for making full use of native and artificial metalloenzymes in environmental science are also prospected. [ABSTRACT FROM AUTHOR]

Protein design is able to create artificial proteins with advanced functions, and computer simulation plays a key role in guiding the rational design. In the absence of structural evidence for cytoglobin (Cgb) with an intramolecular disulfide bond, we recently designed a de novo disulfide bond in myoglobin (Mb) based on structural alignment (i.e., V21C/V66C Mb double mutant). To provide deep insight into the regulation role of the Cys21-Cys66 disulfide bond, we herein perform molecular dynamics (MD) simulation of the fluoride–protein complex by using a fluoride ion as a probe, which reveals detailed interactions of the fluoride ion in the heme distal pocket, involving both the distal His64 and water molecules. Moreover, we determined the kinetic parameters of fluoride binding to the double mutant. The results agree with the MD simulation and show that the formation of the Cys21-Cys66 disulfide bond facilitates both fluoride binding to and dissociating from the heme iron. Therefore, the combination of theoretical and experimental studies provides valuable information for understanding the structure and function of heme proteins, as regulated by a disulfide bond. This study is thus able to guide the rational design of artificial proteins with tunable functions in the future. [ABSTRACT FROM AUTHOR]

Nonenzymatic glycation and the subsequent accumulation of advanced glycation end-products (AGEs) in proteins are factors underlying long-term pathogenesis in diabetes. The study of protein glycation is crucial for elucidating their relationship with diabetes mellitus and related disorders. This study explores the interaction between d -ribose and human myoglobin (HMb), as well as the protective effect of thymoquinone (TQ) on glycation. A time-dependent in-vitro glycation study was performed to investigate the mechanism of d -ribose-induced structural interference of HMb in the absence and presence of TQ. Spectroscopic and proteomic analysis indicated that the presence of TQ significantly reduced the total amount of AGEs while maintaining structural characteristics of HMb. 14 glycated sites on HMb were further identified via liquid chromatography-tandem mass spectrometry (LC-MS/MS) after incubation with d -ribose for 12 h, predominantly interacting with lysine residues. TQ was found to disrupt this interaction, reducing the glycated sites from 14 to 12 sites and the percentage of glycated peptides from 26.50 % to 12.97 %. Additionally, there was a significant decrease in the degree of glycation at the same sites. In summary, our findings suggest that TQ has the potential to act as an anti-glycation agent and provide a comprehensive understanding underlying the inhibition mechanism of glycation. • Inhibition of glycation is crucial in preventing diabetes-related complications. • Thymoquinone exerts a significant inhibitory effect on the d -ribose-induced glycation of human myoglobin. • Thymoquinone inhibits the formation of advanced glycation end products. • The number of glycation sites on human myoglobin is decreased in the presence of thymoquinone. • The study suggests the potential of quinone compounds as effective anti-glycation drugs. [ABSTRACT FROM AUTHOR]

Heme proteins perform a large array of biological functions, with the heme group bound non-covalently or covalently. To probe the stabilization role of conserved tyrosine residue in the secondary sphere of heme site in heme proteins, we herein used cytochrome b 5 (Cyt b 5 ) as a model protein, and mutated Tyr30 to Phe or His by removal of Tyr30 associated H-bond network and hydrophobic interaction. We performed thermal-induced unfolding studies for the two mutants, Y30F Cyt b 5 and Y30H Cyt b 5 , as monitored by both UV–Vis and CD spectroscopy, as well as heme transfer studies from these proteins to apo-myoglobin, with wild-type Cyt b 5 under the same conditions for comparison. The reduced stability of both mutants indicates that both the H-bonding and hydrophobic interactions associated with Tyr30 contribute to the protein stability. Moreover, we performed molecular modeling studies, which revealed that the hydrophobic interaction in the local region of Y30F Cyt b 5 was well-remained, whereas Y30H Cyt b 5 formed an H-bond network. These observations suggest that the conserved Tyr30 in Cyt b 5 is not replaceable due to the presence of both the H-bond network and hydrophobic interaction in the secondary sphere of the heme active site. As demonstrated here for Cyt b 5 , it may be of practical importance for design of artificial heme proteins by engineering a Tyr in the secondary sphere with improved properties and functions. [ABSTRACT FROM AUTHOR]

To reveal the structure-function relationship of heme proteins, and to provide clues for creating artificial heme proteins with improved functions, we here use myoglobin (Mb) as a model protein, and report that its peroxidase activity can be enhanced by construction of two distal histidines and a channel to the heme pocket. It showed that in addition to a single distal histidine with a suitable distance to the heme iron (Phe43 to His43 mutation), a second distal histidine (Leu29 to His29 mutation) can work cooperatively to increase the turnover number, mimicking the role of well-known His-Arg pair in native peroxidases. Moreover, a channel created to the heme pocket by removal of the native His64 gate (His64 to Ala64 mutation) was shown to facilitate the binding of substrate, resulting in enhanced catalytic efficiency for the triple mutant L29H/F43H/H64A Mb, which is beyond the addition of both double mutants, L29H/H64A Mb and F43H/H64A Mb. These results provide valuable information for elucidating the structure-function relationship of heme proteins. In addition, this study provides clues for design of artificial heme proteins, and the strategy of creating a channel to the heme active center is expected be extended to design of other artificial enzymes with improved catalytic performance. [ABSTRACT FROM AUTHOR]

Although heme proteins are extensively studied, the structure and hydrolysis activity relationship of heme proteins is still not well understood. We herein made a comparison of the heme active site of myoglobin (Mb) and its two distal mutants, L29H Mb and L29E Mb, and found that the heme distal hydrogen-bonding network regulates the hydrolysis activity, with L29E Mb being more reactive. Fluoride competitive binding studies revealed that L29E Mb has an axial water molecule readily replaced by fluoride anion, whereas L29H Mb has a much more stable axial water molecule. pH titration studies showed that L29E Mb exhibits a lower p K a and L29H Mb exhibits a higher p K a of the heme coordinated water molecule compared to that of Mb, rationalizing the relative hydrolysis activities. Moreover, pH-jump induced unfolding studies suggested that the heme distal hydrogen-bonding network, especially in L29H Mb, plays a key role in stabilization of the protein. This study thus provides valuable information for understanding the structure and hydrolysis activity relationship of heme proteins, as well as for rational protein design with the knowledge of structural and functional diversity of heme proteins. [ABSTRACT FROM AUTHOR]

Highlights: [•] L29H Mb in DDAB film on electrode oxidizes guaiacol using O2 as an oxidant. [•] L29H Mb has a ∼3.6-fold higher reactivity than that of Mb at pH 7.0. [•] Distal histidines in L29H Mb play a key role in activation of O2 by providing protons. [ABSTRACT FROM AUTHOR]