Structural Determinants of the Specific Activities of an L-Amino Acid Oxidase from Pseudoalteromonas luteoviolacea CPMOR-1 with Broad Substrate Specificity.

The Pseudoalteromonas luteoviolacea strain CPMOR-1 expresses a flavin adenine dinucleotide (FAD)-dependent L-amino acid oxidase (LAAO) with broad substrate specificity. Steady-state kinetic analysis of its reactivity towards the 20 proteinogenic amino acids showed some activity to all except proline. The relative specific activity for amino acid substrates was not correlated only with Km or kcat values, since the two parameters often varied independently of each other. Variation in Km was attributed to the differential binding affinity. Variation in kcat was attributed to differential positioning of the bound substrate relative to FAD that decreased the reaction rate. A structural model of this LAAO was compared with structures of other FAD-dependent LAAOs that have different substrate specificities: an LAAO from snake venom that prefers aromatic amino acid substrates and a fungal LAAO that is specific for lysine. While the amino acid sequences of these LAAOs are not very similar, their overall structures are comparable. The differential activity towards specific amino acids was correlated with specific residues in the active sites of these LAAOs. Residues in the active site that interact with the amino and carboxyl groups attached to the α-carbon of the substrate amino acid are conserved in all of the LAAOs. Residues that interact with the side chains of the amino acid substrates show variation. This provides insight into the structural determinants of the LAAOs that dictate their different substrate preferences. These results are of interest for harnessing these enzymes for possible applications in biotechnology, such as deracemization.

Substitution of the sole tryptophan of the cupredoxin, amicyanin, with 5-hydroxytryptophan alters fluorescence properties and energy transfer to the type 1 copper site.

Amicyanin is a type 1 copper protein with a single tryptophan residue. Using genetic code expansion, the tryptophan was selectively replaced with the unnatural amino acid, 5-hydroxytryptophan (5-HTP). The 5-HTP substituted amicyanin exhibited absorbance at 300-320 nm, characteristic of 5-HTP and not seen in native amicyanin. The fluorescence emission maximum in 5-HTP substituted amicyanin is redshifted from 318 nm in native amicyanin to 331 nm and to 348 nm in the unfolded protein. The fluorescence quantum yield of 5-HTP substituted amicyanin mutant was much less than that of native amicyanin. Differences in intrinsic fluorescence are explained by differences in the excited states of tryptophan versus 5-HTP and the intraprotein environment. The substitution of tryptophan with 5-HTP did not affect the visible absorbance and redox potential of the copper, which is 10 Å away. In amicyanin and other cupredoxins, an unexplained quenching of the intrinsic fluorescence by the bound copper is observed. However, the fluorescence of 5-HTP substituted amicyanin is not quenched by the copper. It is shown that the mechanism of quenching in native amicyanin is Förster, or fluorescence, resonance energy transfer (FRET). This does not occur in 5-HTP substituted amicyanin because the fluorescence quantum yield is significantly lower and the red-shift of fluorescence emission maximum decreases overlap with the near UV absorbance of copper. Characterization of the distinct fluorescence properties of 5-HTP relative to tryptophan in amicyanin provides a basis for spectroscopic interrogation of the protein microenvironment using 5-HTP, and long-distance interactions with transition metals.

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.

Diversity of structures and functions of oxo-bridged non-heme diiron proteins.

Oxo-bridged diiron proteins are a distinct class of non-heme iron proteins. Their active sites are composed of two irons that are coordinated by amino acid side chains, and a bridging oxygen that interacts with each iron. These proteins are members of the ferritin superfamily and share the structural feature of a four α-helix bundle that provides the residues that coordinate the irons. The different proteins also display a wide range of structures and functions. A prototype of this family is hemerythrin, which functions as an oxygen transporter. Several other hemerythrin-like proteins have been described with a diversity of functions including oxygen and iron sensing, and catalytic activities. Rubrerythrins react with hydrogen peroxide and rubrerythrin-like proteins possess a rubredoxin domain, in addition to the oxo-bridged diiron center. Other redox enzymes with oxo-bridged irons include flavodiiron proteins that act as O2 or NO reductases, ribonucleotide reductase and methane monooxygenase. Ferritins have an oxo-bridged diiron in the ferroxidase center of the protein, which plays a role in the iron storage function of these proteins. There are also bacterial ferritins that exhibit catalytic activities. The structures and functions of this broad class of oxo-bridged diiron proteins are described and compared in this review.

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.

Roles of active-site residues in catalysis, substrate binding, cooperativity, and the reaction mechanism of the quinoprotein glycine oxidase.

The quinoprotein glycine oxidase from the marine bacterium Pseudoalteromonas luteoviolacea (PlGoxA) uses a protein-derived cysteine tryptophylquinone (CTQ) cofactor to catalyze conversion of glycine to glyoxylate and ammonia. This homotetrameric enzyme exhibits strong cooperativity toward glycine binding. It is a good model for studying enzyme kinetics and cooperativity, specifically for being able to separate those aspects of protein function through directed mutagenesis. Variant proteins were generated with mutations in four active-site residues, Phe-316, His-583, Tyr-766, and His-767. Structures for glycine-soaked crystals were obtained for each. Different mutations had differential effects on kcat and K0.5 for catalysis, K0.5 for substrate binding, and the Hill coefficients describing the steady-state kinetics or substrate binding. Phe-316 and Tyr-766 variants retained catalytic activity, albeit with altered kinetics and cooperativity. Substitutions of His-583 revealed that it is essential for glycine binding, and the structure of H583C PlGoxA had no active-site glycine present in glycine-soaked crystals. The structure of H767A PlGoxA revealed a previously undetected reaction intermediate, a carbinolamine product-reduced CTQ adduct, and exhibited only negligible activity. The results of these experiments, as well as those with the native enzyme and previous variants, enabled construction of a detailed mechanism for the reductive half-reaction of glycine oxidation. This proposed mechanism includes three discrete reaction intermediates that are covalently bound to CTQ during the reaction, two of which have now been structurally characterized by X-ray crystallography.

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.

Kinetic and structural evidence that Asp-678 plays multiple roles in catalysis by the quinoprotein glycine oxidase.

PlGoxA from Pseudoalteromonas luteoviolacea is a glycine oxidase that utilizes a protein-derived cysteine tryptophylquinone (CTQ) cofactor. A notable feature of its catalytic mechanism is that it forms a stable product-reduced CTQ adduct that is not hydrolyzed in the absence of O2 Asp-678 resides near the quinone moiety of PlGoxA, and an Asp is structurally conserved in this position in all tryptophylquinone enzymes. In those other enzymes, mutation of that Asp results in no or negligible CTQ formation. In this study, mutation of Asp-678 in PlGoxA did not abolish CTQ formation. This allowed, for the first time, studying the role of this residue in catalysis. D678A and D678N substitutions yielded enzyme variants with CTQ, which did not react with glycine, although glycine was present in the crystal structures in the active site. D678E PlGoxA was active but exhibited a much slower kcat This mutation altered the kinetic mechanism of the reductive half-reaction such that one could observe a previously undetected reactive intermediate, an initial substrate-oxidized CTQ adduct, which converted to the product-reduced CTQ adduct. These results indicate that Asp-678 is involved in the initial deprotonation of the amino group of glycine, enabling nucleophilic attack of CTQ, as well as the deprotonation of the substrate-oxidized CTQ adduct, which is coupled to CTQ reduction. The structures also suggest that Asp-678 is acting as a proton relay that directs these protons to a water channel that connects the active sites on the subunits of this homotetrameric enzyme.

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.

Structural and Spectroscopic Characterization of a Product Schiff Base Intermediate in the Reaction of the Quinoprotein Glycine Oxidase, GoxA.

The LodA-like proteins make up a recently identified family of enzymes that rely on a cysteine tryptophylquinone cofactor for catalysis. They differ from other tryptophylquinone enzymes in that they are oxidases rather than dehydrogenases. GoxA is a member of this family that catalyzes the oxidative deamination of glycine. Our previous work with GoxA from Pseudoalteromonas luteoviolacea demonstrated that this protein forms a stable intermediate upon anaerobic incubation with glycine. The spectroscopic properties of this species were unique among those identified for tryptophylquinone enzymes characterized to date. Here we use X-ray crystallography and resonance Raman spectroscopy to identify the GoxA catalytic intermediate as a product Schiff base. Structural work additionally highlights features of the active site pocket that confer substrate specificity, intermediate stabilization, and catalytic activity. The unusual properties of GoxA are discussed within the context of the other tryptophylquinone enzymes.

Diversity of structures, catalytic mechanisms and processes of cofactor biosynthesis of tryptophylquinone-bearing enzymes.

Tryptophyquinone-bearing enzymes contain protein-derived cofactors formed by posttranslational modifications of Trp residues. Tryptophan tryptophylquinone (TTQ) is comprised of a di-oxygenated Trp residue, which is cross-linked to another Trp residue. Cysteine tryptophylquinone (CTQ) is comprised of a di-oxygenated Trp residue, which is cross-linked to a Cys residue. Despite the similarity of these cofactors, it has become evident in recent years that the overall structures of the enzymes that possess these cofactors vary, and that the gene clusters that encode the enzymes are quite diverse. While it had been long assumed that all tryptophylquinone enzymes were dehydrogenases, recently discovered classes of these enzymes are oxidases. A common feature of enzymes that have these cofactors is that the posttranslational modifications that form the mature cofactors are catalyzed by a modifying enzyme. However, it is now clear that modifying enzymes are different for different tryptophylquinone enzymes. For methylamine dehydrogenase a di-heme enzyme, MauG, is needed to catalyze TTQ biosynthesis. However, no gene similar to mauG is present in the gene clusters that encode the other enzymes, and the recently characterized family of CTQ-dependent oxidases, termed LodA-like proteins, require a flavoenzyme for cofactor biosynthesis.

Metabolomics reveals critical adrenergic regulatory checkpoints in glycolysis and pentose-phosphate pathways in embryonic heart.

Cardiac energy demands during early embryonic periods are sufficiently met through glycolysis, but as development proceeds, the oxidative phosphorylation in mitochondria becomes increasingly vital. Adrenergic hormones are known to stimulate metabolism in adult mammals and are essential for embryonic development, but relatively little is known about their effects on metabolism in the embryonic heart. Here, we show that embryos lacking adrenergic stimulation have ∼10-fold less cardiac ATP compared with littermate controls. Despite this deficit in steady-state ATP, neither the rates of ATP formation nor degradation was affected in adrenergic hormone-deficient hearts, suggesting that ATP synthesis and hydrolysis mechanisms were fully operational. We thus hypothesized that adrenergic hormones stimulate metabolism of glucose to provide chemical substrates for oxidation in mitochondria. To test this hypothesis, we employed a metabolomics-based approach using LC/MS. Our results showed glucose 1-phosphate and glucose 6-phosphate concentrations were not significantly altered, but several downstream metabolites in both glycolytic and pentose-phosphate pathways were significantly lower compared with controls. Furthermore, we identified glyceraldehyde-3-phosphate dehydrogenase and glucose-6-phosphate dehydrogenase as key enzymes in those respective metabolic pathways whose activity was significantly (p < 0.05) and substantially (80 and 40%, respectively) lower in adrenergic hormone-deficient hearts. Addition of pyruvate and to a lesser extent ribose led to significant recovery of steady-state ATP concentrations. These results demonstrate that without adrenergic stimulation, glucose metabolism in the embryonic heart is severely impaired in multiple pathways, ultimately leading to insufficient metabolic substrate availability for successful transition to aerobic respiration needed for survival.

Protein-Derived Cofactors Revisited: Empowering Amino Acid Residues with New Functions.

A protein-derived cofactor is a catalytic or redox-active site in a protein that is formed by post-translational modification of one or more amino acid residues. These post-translational modifications are irreversible and endow the modified amino acid residues with new functional properties. This Perspective focuses on the following advances in this area that have occurred during recent years. The biosynthesis of the tryptophan tryptophylquinone cofactor is catalyzed by a diheme enzyme, MauG. A bis-FeIV redox state of the hemes performs three two-electron oxidations of specific Trp residues via long-range electron transfer. In contrast, a flavoenzyme catalyzes the biosynthesis of the cysteine tryptophylquinone (CTQ) cofactor present in a newly discovered family of CTQ-dependent oxidases. Another carbonyl cofactor, the pyruvoyl cofactor found in classes of decarboxylases and reductases, is formed during an apparently autocatalytic cleavage of a precursor protein at the N-terminus of the cleavage product. It has been shown that in at least some cases, the cleavage is facilitated by binding to an accessory protein. Tyrosylquinonine cofactors, topaquinone and lysine tyrosylquinone, are found in copper-containing amine oxidases and lysyl oxidases, respectively. The physiological roles of different families of these enzymes in humans have been more clearly defined and shown to have significant implications with respect to human health. There has also been continued characterization of the roles of covalently cross-linked amino acid side chains that influence the reactivity of redox-active metal centers in proteins. These include Cys-Tyr species in galactose oxidase and cysteine dioxygenase and the Met-Tyr-Trp species in the catalase-peroxidase KatG.

Structure and Enzymatic Properties of an Unusual Cysteine Tryptophylquinone-Dependent Glycine Oxidase from Pseudoalteromonas luteoviolacea.

Glycine oxidase from Pseudoalteromonas luteoviolacea (PlGoxA) is a cysteine tryptophylquinone (CTQ)-dependent enzyme. Sequence analysis and phylogenetic analysis place it in a newly designated subgroup (group IID) of a recently identified family of LodA-like proteins, which are predicted to possess CTQ. The crystal structure of PlGoxA reveals that it is a homotetramer. It possesses an N-terminal domain with no close structural homologues in the Protein Data Bank. The active site is quite small because of intersubunit interactions, which may account for the observed cooperativy toward glycine. Steady-state kinetic analysis yielded the following values: kcat = 6.0 ± 0.2 s-1, K0.5 = 187 ± 18 µM, and h = 1.77 ± 0.27. In contrast to other quinoprotein amine dehydrogenases and oxidases that exhibit anomalously large primary kinetic isotope effects on the rate of reduction of the quinone cofactor by the amine substrate, no significant primary kinetic isotope effect was observed for this reaction of PlGoxA. The absorbance spectrum of glycine-reduced PlGoxA exhibits features in the range of 400-650 nm that have not previously been seen in other quinoproteins. Thus, in addition to the unusual structural features of PlGoxA, the kinetic and chemical reaction mechanisms of the reductive half-reaction of PlGoxA appear to be distinct from those of other amine dehydrogenases and amine oxidases that use tryptophylquinone and tyrosylquinone cofactors.

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.

Roles of Copper and a Conserved Aspartic Acid in the Autocatalytic Hydroxylation of a Specific Tryptophan Residue during Cysteine Tryptophylquinone Biogenesis.

The first posttranslational modification step in the biosynthesis of the tryptophan-derived quinone cofactors is the autocatalytic hydroxylation of a specific Trp residue at position C-7 on the indole side chain. Subsequent modifications are catalyzed by modifying enzymes, but the mechanism by which this first step occurs is unknown. LodA possesses a cysteine tryptophylquinone (CTQ) cofactor. Metal analysis as well as spectroscopic and kinetic studies of the mature and precursor forms of a D512A LodA variant provides evidence that copper is required for the initial hydroxylation of the precursor protein and that if alternative metals are bound, the modification does not occur and the precursor is unstable. It is shown that the mature native LodA also contains loosely bound copper, which affects the visible absorbance spectrum and quenches the fluorescence spectrum that is attributed to the mature CTQ cofactor. When copper is removed, the fluorescence appears, and when it is added back to the protein, the fluorescence is quenched, indicating that copper reversibly binds in the proximity of CTQ. Removal of copper does not diminish the enzymatic activity of LodA. This distinguishes LodA from enzymes with protein-derived tyrosylquinone cofactors in which copper is present near the cofactor and is absolutely required for activity. Mechanisms are proposed for the role of copper in the hydroxylation of the unactivated Trp side chain. These results demonstrate that the reason that the highly conserved Asp512 is critical for LodA, and possibly all tryptophylquinone enzymes, is not because it is required for catalysis but because it is necessary for CTQ biosynthesis, more specifically to facilitate the initial copper-dependent hydroxylation of a specific Trp residue.

In Silico Approaches to Identify Mutagenesis Targets to Probe and Alter Protein-Cofactor and Protein-Protein Functional Relationships.

When performing site-directed mutagenesis experiments to study protein structure-function relationships, ideally one would know the structure of the protein under study. It is also very useful to have structures of multiple related proteins in order to determine whether or not particular amino acid residues are conserved in the structures either in the active site of an enzyme at the surface of a protein or at a putative protein-protein interface. While many protein structures are available in the Protein Data Base (PDB), a structure of the protein of interest may not be available. In the study of reversible and often transient protein-protein interactions it is rare to have a structure of the complex of the two interacting proteins. In this chapter, methods are described for comparing protein structures, generating putative structures of proteins with homology models based on the protein primary sequence, and generating docking models to predict interaction sites between proteins and cofactor-protein interactions. The rationale used to predict mutagenesis targets from these structures and models is also described.