The hemerythrin-like diiron protein from Mycobacterium kansasii is a nitric oxide peroxidase.

The hemerythrin-like protein from Mycobacterium kansasii (Mka HLP) is a member of a distinct class of oxo-bridged diiron proteins that are found only in mycobacterial species that cause respiratory disorders in humans. Because it had been shown to exhibit weak catalase activity and a change in absorbance on exposure to nitric oxide (NO), the reactivity of Mka HLP toward NO was examined under a variety of conditions. Under anaerobic conditions, we found that NO was converted to nitrite (NO2-) via an intermediate, which absorbed light at 520 nm. Under aerobic conditions NO was converted to nitrate (NO3-). In each of these two cases, the maximum amount of nitrite or nitrate formed was at best stoichiometric with the concentration of Mka HLP. When incubated with NO and H2O2, we observed NO peroxidase activity yielding nitrite and water as reaction products. Steady-state kinetic analysis of NO consumption during this reaction yielded a Km for NO of 0.44 µM and a kcat/Km of 2.3 × 105 M-1s-1. This high affinity for NO is consistent with a physiological role for Mka HLP in deterring nitrosative stress. This is the first example of a peroxidase that uses an oxo-bridged diiron center and a rare example of a peroxidase utilizing NO as an electron donor and cosubstrate. This activity provides a mechanism by which the infectious Mycobacterium may combat against the cocktail of NO and superoxide (O2•-) generated by macrophages to defend against bacteria, as well as to produce NO2- to adapt to hypoxic conditions.

Correlation of Conservation of Sequence and Structures of Mycobacterial Hemerythrin-like Proteins with Evolutionary Relationship and Host Pathogenicity.

The Rv2633c gene of Mycobacterium tuberculosis, which plays a role in infection, encodes a hemerythrin-like protein (HLP). The crystal structure of an orthologue of Rv2633c, the HLP from Mycobacterium kansasii, revealed that it possessed structural features that were distinct from other hemerythrins and HLPs. These and other orthologous proteins comprise a distinct class of non-heme di-iron HLPs that are only found in mycobacteria. This study presents an analysis and comparison of protein sequences, putative structures, and evolutionary relationship of HLPs from 20 mycobacterial species that are known to cause tuberculosis or pulmonary disorders in humans. The results of this analysis allowed correlation of the physicochemical characteristics of amino acid residues that are substituted in these highly conserved sequences with their position in structures, possible effects on function, and evolutionary relationships. The sequences of the proteins from M. tuberculosis, Mycobacterium bovis, and other members of the M. tuberculosis complex, which cause tuberculosis, have substitutions not seen in the other non-tuberculous mycobacteria. Furthermore, groups of species that are closely related, based on phylogenetic analysis, possess substitutions of otherwise conserved residues not seen in other species that are less related. This information is correlated with the occurrence and clinical presentations of these groups of mycobacterial species. The results of this study provide a framework for structure-function studies to determine how subtle differences in the primary sequences of members of this family of proteins correlate with their structures and activities and how this may influence the infectious properties of the host species.

Crystal structure of a hemerythrin-like protein from Mycobacterium kansasii and homology model of the orthologous Rv2633c protein of M. tuberculosis.

Pathogenic and opportunistic mycobacteria have a distinct class of non-heme di-iron hemerythrin-like proteins (HLPs). The first to be isolated was the Rv2633c protein, which plays a role in infection by Mycobacterium tuberculosis (Mtb), but could not be crystallized. This work presents the first crystal structure of an ortholog of Rv2633c, the mycobacterial HLP from Mycobacterium kansasii (Mka). This structure differs from those of hemerythrins and other known HLPs. It consists of five α-helices, whereas all other HLP domains have four. In contrast with other HLPs, the HLP domain is not fused to an additional protein domain. The residues ligating and surrounding the di-iron site are also unique among HLPs. Notably, a tyrosine occupies the position normally held by one of the histidine ligands in hemerythrin. This structure was used to construct a homology model of Rv2633c. The structure of five α-helices is conserved and the di-iron site ligands are identical in Rv2633c. Two residues near the ends of helices in the Mka HLP structure are replaced with prolines in the Rv2633c model. This may account for structural perturbations that decrease the solubility of Rv2633c relative to Mka HLP. Clusters of residues that differ in charge or polarity between Rv2633c and Mka HLP that point outward from the helical core could reflect a specificity for potential differential interactions with other protein partners in vivo, which are related to function. The Mka HLP exhibited weaker catalase activity than Rv2633c. Evidence was obtained for the interaction of Mka HLP irons with nitric oxide.

Characterization of PlGoxB, a flavoprotein required for cysteine tryptophylquinone biosynthesis in glycine oxidase from Pseudoalteromonas luteoviolacea.

LodA-like proteins are oxidases with a protein-derived cysteine tryptophylquinone (CTQ) prosthetic group. In Pseudoalteromonas luteoviolacea glycine oxidase (PlGoxA), CTQ biosynthesis requires post-translational modifications catalyzed by a modifying enzyme encoded by PlgoxB. The PlGoxB protein was expressed and shown to possess a flavin cofactor. PlGoxB was unstable in solution as it readily lost the flavin and precipitated. PlGoxB precipitation was significantly reduced by incubation with either excess FAD or an equal concentration of prePlGoxA, the precursor protein that is its substrate. In contrast, the mature CTQ-bearing PlGoxA had no stabilizing effect. A homology model of PlGoxB was generated using the structure of Alkylhalidase CmIS. The FAD-binding site of PlGoxB in the model was nearly identical to that of the template structure. The bound FAD in PlGoxB had significant solvent exposure, consistent with the observed tendency to lose FAD. This also suggested that interaction of prePlGoxA with PlGoxB at the exposed FAD-binding site could prevent the observed loss of FAD and subsequent precipitation of PlGoxB. A docking model of the putative PlGoxB-prePlGoxA complex was consistent with these hypotheses. The experimental results and computational analysis implicate structural features of PlGoxB that contribute to its stability and function.

The Redox Properties of a Cysteine Tryptophylquinone-Dependent Glycine Oxidase Are Distinct from Those of Tryptophylquinone-Dependent Dehydrogenases.

GoxA is a cysteine tryptophylquinone (CTQ)-dependent glycine oxidase that is a member of a family of LodA-like proteins. The electrochemical midpoint potential ( Em) values for the quinone/semiquinone couple and the semiquinone/quinol couple were determined to be 111 and 21, respectively. The Em value for the overall two-electron quinone/quinol couple was similar to those of CTQ- and tryptophan tryptophylquinone (TTQ)-bearing dehydrogenases. However, for the well-studied TTQ-dependent methylamine dehydrogenase, the quinone/semiquinone couple is more negative than the semiquinone/quinol couple, the opposite of what was determined for GoxA. The change in Em value for the two-electron quinone/quinol couple of CTQ in GoxA with pH indicates that the overall two-electron transfer process is associated with the transfer of one proton. Thus, the quinol is anionic. The data reported herein further suggest that in GoxA the CTQ semiquinone is neutral, in contrast to the TTQ-dependent dehydrogenases, in which it is an anionic TTQ semiquinone. These results are discussed in the context of the structure and function of this glycine oxidase, compared to that of the tryptophylquinone-dependent dehydrogenases.

The Rv2633c protein of Mycobacterium tuberculosis is a non-heme di-iron catalase with a possible role in defenses against oxidative stress.

The Rv2633c gene in Mycobacterium tuberculosis is rapidly up-regulated after macrophage infection, suggesting that Rv2633c is involved in M. tuberculosis pathogenesis. However, the activity and role of the Rv2633c protein in host colonization is unknown. Here, we analyzed the Rv2633c protein sequence, which revealed the presence of an HHE cation-binding domain common in hemerythrin-like proteins. Phylogenetic analysis indicated that Rv2633c is a member of a distinct subset of hemerythrin-like proteins exclusive to mycobacteria. The Rv2633c sequence was significantly similar to protein sequences from other pathogenic strains within that subset, suggesting that these proteins are involved in mycobacteria virulence. We expressed and purified the Rv2633c protein in Escherichia coli and found that it contains two iron atoms, but does not behave like a hemerythrin. It migrated as a dimeric protein during size-exclusion chromatography. It was not possible to reduce the protein or observe any evidence for its interaction with O2 However, Rv2633c did exhibit catalase activity with a kcat of 1475 s-1 and Km of 10.1 ± 1.7 mm Cyanide and azide inhibited the catalase activity with Ki values of 3.8 µm and 37.7 µm, respectively. Rv2633c's activity was consistent with a role in defenses against oxidative stress generated during host immune responses after M. tuberculosis infection of macrophages. We note that Rv2633c is the first example of a non-heme di-iron catalase, and conclude that it is a member of a subset of hemerythrin-like proteins exclusive to mycobacteria, with likely roles in protection against host defenses.

Ascorbate protects the diheme enzyme, MauG, against self-inflicted oxidative damage by an unusual antioxidant mechanism.

Ascorbate protects MauG from self-inactivation that occurs during the autoreduction of the reactive bis-FeIV state of its diheme cofactor. The mechanism of protection does not involve direct reaction with reactive oxygen species in solution. Instead, it binds to MauG and mitigates oxidative damage that occurs via internal transfer of electrons from amino acid residues within the protein to the high-valent hemes. The presence of ascorbate does not inhibit the natural catalytic reaction of MauG, which catalyzes oxidative post-translational modifications of a substrate protein that binds to the surface of MauG and is oxidized by the high-valent hemes via long-range electron transfer. Ascorbate was also shown to prolong the activity of a P107V MauG variant that is more prone to inactivation. A previously unknown ascorbate peroxidase activity of MauG was characterized with a kcat of 0.24 s-1 and a Km of 2.2 µM for ascorbate. A putative binding site for ascorbate was inferred from inspection of the crystal structure of MauG and comparison with the structure of soybean ascorbate peroxidase with bound ascorbate. The ascorbate bound to MauG was shown to accelerate the rates of both electron transfers to the hemes and proton transfers to hemes which occur during the multistep autoreduction to the diferric state which is accompanied by oxidative damage. A structural basis for these effects is inferred from the putative ascorbate-binding site. This could be a previously unrecognized mechanism by which ascorbate mitigates oxidative damage to heme-dependent enzymes and redox proteins in nature.

Properties of the high-spin heme of MauG are altered by binding of preMADH at the protein surface 40 Å away.

The diheme enzyme MauG catalyzes oxidative post-translational modifications of a protein substrate, precursor protein of methylamine dehydrogenase (preMADH), that binds to the surface of MauG. The high-spin heme iron of MauG is located 40 Å from preMADH. The ferric heme is an equilibrium of five- and six-coordinate states. PreMADH binding increases the proportion of five-coordinate heme three-fold. On reaction of MauG with H2 O2 both hemes become FeIV . In the absence of preMADH the hemes autoreduce to ferric in a multistep process involving multiple electron and proton transfers. Binding of preMADH in the absence of catalysis alters the mechanism of autoreduction of the ferryl heme. Thus, substrate binding alters the environment in the distal heme pocket of the high-spin heme over very long distance.

A Suicide Mutation Affecting Proton Transfers to High-Valent Hemes Causes Inactivation of MauG during Catalysis.

In the absence of its substrate, the autoreduction of the high-valent bis-FeIV state of the hemes of MauG to the diferric state proceeds via a Compound I-like and then a Compound II-like intermediate. This process is coupled to oxidative damage to specific methionine residues and inactivation of MauG. The autoreduction of a P107V MauG variant, which is more prone to oxidative damage, proceeds directly from the bis-FeIV to the Compound II-like state with no detectable Compound I intermediate. Comparison of the crystal structures of native and P107V MauG reveals that this mutation alters the positions of amino acid residues in the heme site as well as the water network that delivers protons from the solvent to the hemes during their reduction. Kinetic, spectroscopic, and solvent kinetic isotope effect studies demonstrate that these changes in the heme site affect the protonation state of the ferryl heme and the relative efficiencies of two alternative pathways for the transfer of protons from solvent to the hemes. These changes enhance the rate of autoreduction of P107V MauG such that it competes with the catalytic reaction with substrate and causes the enzyme to inactivate itself during the steady-state reaction with H2O2 and its substrate. Thus, while this mutation has negligible effects on the initial steady-state kinetic parameters of MauG, it is a fatal mutation as it causes inactivation during catalysis.

Mechanism of protein oxidative damage that is coupled to long-range electron transfer to high-valent haems.

In the absence of its substrate, the auto-reduction of the high-valent bis-Fe(IV) state of the dihaem enzyme MauG is coupled to oxidative damage of a methionine residue. Transient kinetic and solvent isotope effect studies reveal that this process occurs via two sequential long-range electron transfer (ET) reactions from methionine to the haems. The first ET is coupled to proton transfer (PT) to the haems from solvent via an ordered water network. The second ET is coupled to PT at the methionine site and occurs during the oxidation of the methionine to a sulfoxide. This process proceeds via Compound I- and Compound II-like haem intermediates. It is proposed that the methionine radical is stabilized by a two-centre three-electron (2c3e) bond. This provides insight into how oxidative damage to proteins may occur without direct contact with a reactive oxygen species, and how that damage can be propagated through the protein.

Roles of multiple-proton transfer pathways and proton-coupled electron transfer in the reactivity of the bis-FeIV state of MauG.

The high-valent state of the diheme enzyme MauG exhibits charge-resonance (CR) stabilization in which the major species is a bis-Fe(IV) state with one heme present as Fe(IV)=O and the other as Fe(IV) with axial heme ligands provided by His and Tyr side chains. In the absence of its substrate, the high-valent state is relatively stable and returns to the diferric state over several minutes. It is shown that this process occurs in two phases. The first phase is redistribution of the resonance species that support the CR. The second phase is the loss of CR and reduction to the diferric state. Thermodynamic analysis revealed that the rates of the two phases exhibited different temperature dependencies and activation energies of 8.9 and 19.6 kcal/mol. The two phases exhibited kinetic solvent isotope effects of 2.5 and 2.3. Proton inventory plots of each reaction phase exhibited extreme curvature that could not be fit to models for one- or multiple-proton transfers in the transition state. Each did fit well to a model for two alternative pathways for proton transfer, each involving multiple protons. In each case the experimentally determined fractionation factors were consistent with one of the pathways involving tunneling. The percent of the reaction that involved the tunneling pathway differed for the two reaction phases. Using the crystal structure of MauG it was possible to propose proton-transfer pathways consistent with the experimental data using water molecules and amino acid side chains in the distal pocket of the high-spin heme.