Functional roles of the 6-S-cysteinyl, 8alpha-N1-histidyl FAD in glucooligosaccharide oxidase from Acremonium strictum

The crystal structure of glucooligosaccharide oxidase from Acremonium strictum was demonstrated to contain a bicovalent flavinylation, with the 6- and 8alpha-positions of the flavin isoalloxazine ring cross-linked to Cys(130) and His(70), respectively. The H70A and C130A single mutants still retain the covalent FAD, indicating that flavinylation at these two residues is independent. Both mutants exhibit a decreased midpoint potential of approximately +69 and +61 mV, respectively, compared with +126 mV for the wild type, and possess lower activities with k(cat) values reduced to approximately 2 and 5%, and the flavin reduction rate reduced to 0.6 and 14%. This indicates that both covalent linkages increase the flavin redox potential and alter the redox properties to promote catalytic efficiency. In addition, the isolated H70A/C130A double mutant does not contain FAD, and addition of exogenous FAD was not able to restore any detectable activity. This demonstrates that the covalent attachment is essential for the binding of the oxidized cofactor. Furthermore, the crystal structure of the C130A mutant displays conformational changes in several cofactor and substrate-interacting residues and hence provides direct evidence for novel functions of flavinylation in assistance of cofactor and substrate binding. Finally, the wild-type enzyme is more heat and guanidine HCl-resistant than the mutants. Therefore, the bicovalent flavin linkage not only tunes the redox potential and contributes to cofactor and substrate binding but also increases structural stability.     

Interaction of heme with variants of the heme chaperone CcmE carrying active site mutations and a cleavable N-terminal His tag

Cytochrome c maturation in the periplasms of many bacteria requires the heme chaperone CcmE, which binds heme covalently both in vivo and in vitro via a histidine residue before transferring the heme to apocytochromes c. To investigate the mechanism and specificity of heme attachment to CcmE, we have mutated the conserved histidine 130 of a soluble C-terminally His-tagged version of CcmE (CcmEsol-C-His6) from Escherichia coli to alanine or cysteine. Remarkably, covalent bond formation with heme occurs with the protein carrying the cysteine mutation, and the process occurs both in vivo and in vitro. The yield of holo-H130C CcmEsol-C-His6 produced in vivo is low compared with the wild type. In vitro heme attachment occurs only under reducing conditions. We demonstrate the involvement of one of the heme vinyl groups and a side chain at residue 130 in the bond formation by showing that in vitro attachment does not occur either with the heme analogue mesoheme or when alanine is present at residue 130. These results have implications for the mechanism of heme attachment to the histidine of CcmE. In vitro, CcmEsol lacking a His tag binds 8-anilino-1-naphthalenesulphonate and heme, the latter both noncovalently and via a covalent bond from the histidine side chain, similarly to the tagged proteins, thus countering a recent proposal that the His tag causes the heme binding. However, the His tag does appear to enhance the rate of in vitro covalent heme binding and to affect the heme ligation in the ferric b-type cytochrome form.     

6-S-cysteinylation of bi-covalently attached FAD in berberine bridge enzyme tunes the redox potential for optimal activity

A mutagenic analysis of the amino acid residues His-104 and Cys-166, which are involved in the bi-covalent attachment of FAD to berberine bridge enzyme, was performed. Here we present a detailed biochemical characterization of the cysteine link to FAD observed in this recently discovered group of flavoproteins. The C166A mutant protein still has residual activity, but reduced to approximately 6% of the turnover rate observed for wild-type berberine bridge enzyme. A more detailed analysis of single reaction steps by stopped-flow spectrophotometry showed that the reductive half-reaction is greatly influenced by the lack of the 6-S-cysteinyl linkage, resulting in a 370-fold decrease in the rate of flavin reduction. Determination of the redox potentials for both wild type and the C166A mutein revealed that the difference in the redox potential observed can fully account for the change in the kinetic properties. The wild-type protein exhibits a midpoint potential of +132 mV, which is the highest redox potential determined for any flavoenzyme so far. Removal of the cysteine linkage to FAD in the C166A mutein leads to a redox potential of +53 mV, which is in the expected range for flavoproteins with a single covalent attachment of FAD to a His residue via its 8-alpha position. We also show that the biochemical properties of the mutein resemble that of typical flavoprotein oxidases and that deviations from this behavior observed for the wild type are due to the FAD-6-S-cysteinyl bond. In addition, rapid reaction stopped-flow experiments give no indication for a radical mechanism supporting the direct transfer of a hydride from the substrate to the cofactor.     

Glycine oxidase from Bacillus subtilis. Characterization of a new flavoprotein.

Glycine oxidase (GO) is a homotetrameric flavoenzyme that contains one molecule of non-covalently bound flavin adenine dinucleotide per 47 kDa protein monomer. GO is active on various amines (sarcosine, N-ethylglycine, glycine) and d-amino acids (d-alanine, d-proline). The products of GO reaction with various substrates have been determined, and it has been clearly shown that GO catalyzes the oxidative deamination of primary and secondary amines, a reaction similar to that of d-amino acid oxidase, although its sequence homology is higher with enzymes such as sarcosine oxidase and N-methyltryptophane oxidase. GO shows properties that are characteristic of the oxidase class of flavoproteins: it stabilizes the anionic flavin semiquinone and forms a reversible covalent flavin-sulfite complex. The approximately 300 mV separation between the two FAD redox potentials is in accordance with the high amount of the anionic semiquinone formed on photoreduction. GO can be distinguished from d-amino acid oxidase by its low catalytic efficiency and high apparent K(m) value for d-alanine. A number of active site ligands have been identified; the tightest binding is observed with glycolate, which acts as a competitive inhibitor with respect to sarcosine. The presence of a carboxylic group and an amino group on the substrate molecule is not mandatory for binding and catalysis.     

Bacterial defenses against oxidants: mechanistic features of cysteine-based peroxidases and their flavoprotein reductases

Antioxidant defenses include a group of ubiquitous, non-heme peroxidases, designated the peroxiredoxins, which rely on an activated cysteine residue at their active site to catalyze the reduction of hydrogen peroxide, organic hydroperoxides, and peroxynitrite. In the typical 2-Cys peroxiredoxins, a second cysteinyl residue, termed the resolving cysteine, is also involved in intersubunit disulfide bond formation during the course of catalysis by these enzymes. Many bacteria also express a flavoprotein, AhpF, which acts as a dedicated disulfide reductase to recycle the bacterial peroxiredoxin, AhpC, during catalysis. Mechanistic and structural studies of these bacterial proteins have shed light on the linkage between redox state, oligomeric state, and peroxidase activity for the peroxiredoxins, and on the conformational changes accompanying catalysis by both proteins. In addition, these studies have highlighted the dual roles that the oxidized cysteinyl species, cysteine sulfenic acid, can play in eukaryotic peroxiredoxins, acting as a catalytic intermediate in the peroxidase activity, and as a redox sensor in regulating hydrogen peroxide-mediated cell signaling.     

The growing VAO flavoprotein family

The VAO flavoprotein family is a rapidly growing family of oxidoreductases that favor the covalent binding of the FAD cofactor. In this review we report on the catalytic properties of some newly discovered VAO family members and their mode of flavin binding. Covalent binding of the flavin is a self-catalytic post-translational modification primarily taking place in oxidases. Covalent flavinylation increases the redox potential of the cofactor and thus its oxidation power. Recent findings have revealed that some members of the VAO family anchor the flavin via a dual covalent linkage (6-S-cysteinyl-8α-N1-histidyl FAD). Some VAO-type aldonolactone oxidoreductases favor the non-covalent binding of the flavin cofactor. These enzymes act as dehydrogenases, using cytochrome c as electron acceptor.     

Structural characterization of glucooligosaccharide oxidase from Acremonium strictum

Glucooligosaccharide oxidase from Acremonium strictum was screened for potential applications in oligosaccharide acid production and carbohydrate detection. This protein is a unique covalent flavoenzyme which catalyzes the oxidation of a variety of carbohydrates with high selectivity for cello- and maltooligosaccharides. Kinetic measurements suggested that this enzyme possesses an open carbohydrate-binding groove, which is mainly composed of two glucosyl-binding subsites. The encoding gene was subsequently cloned, and one intron was detected in the genomic DNA. Large amounts of active enzymes were expressed in Pichia pastoris, with a yield of 300 mg per liter medium. The protein was predicted to share structural homology with plant cytokinin dehydrogenase and related flavoproteins that share a conserved flavin adenine dinucleotide (FAD)-binding domain. The closest sequence matches are those of plant berberine bridge enzyme-like proteins, particularly the characteristic flavinylation site. Unexpectedly, mutation of the putative FAD-attaching residue, H70, to alanine, serine, cysteine, and tyrosine did not abolish the covalent FAD linkage and had little effect on the Km. Instead, the variants displayed kcat values that were 50- to 600-fold lower, indicating that H70 is crucial for efficient redox catalysis, perhaps through modulation of the oxidative power of the flavin.     

Lipases provide a new mechanistic model for polyhydroxybutyrate (PHB) synthases: characterization of the functional residues in Chromatium vinosum PHB synthase

Polyhydroxybutyrate (PHB) synthases catalyze the conversion of beta-hydroxybutyryl coenzyme A (HBCoA) to PHB. These enzymes require an active site cysteine nucleophile for covalent catalysis. A protein BLASTp search using the Class III Chromatium vinosum synthase sequence reveals high homology to prokaryotic lipases whose crystal structures are known. The homology is very convincing in the alpha-beta-elbow (with the active site nucleophile)-alpha-beta structure, residues 131-175 of the synthase. A conserved histidine of the Class III PHB synthases aligns with the active site histidine of the lipases using the ClustalW algorithm. This is intriguing as this histidine is approximately 200 amino acids removed in sequence space from the catalytic nucleophile. Different threading algorithms suggest that the Class III synthases belong to the alpha/beta hydrolase superfamily which includes prokaryotic lipases. Mutagenesis studies were carried out on C. vinosum synthase C149, H331, H303, D302, and C130 residues. These studies reveal that H331 is the general base catalyst that activates the nucleophile, C149, for covalent catalysis. The model indicates that C130 is not involved in catalysis as previously proposed [Müh, U., Sinskey, A. J., Kirby, D. P., Lane, W. S., and Stubbe, J. (1999) Biochemistry 38, 826-837]. Studies with D302 mutants suggest D302 functions as a general base catalyst in activation of the 3-hydroxyl of HBCoA (or a hydroxybutyrate acyl enzyme) for nucleophilic attack on the covalently linked thiol ester intermediate. The relationship of the lipase model to previous models based on fatty acid synthases is discussed.     

Iron-sulfur flavoprotein (Isf) from Methanosarcina thermophila is the prototype of a widely distributed family

A total of 35 homologs of the iron-sulfur flavoprotein (Isf) from Methanosarcina thermophila were identified in databases. All three domains were represented, and multiple homologs were present in several species. An unusually compact cysteine motif ligating the 4Fe-4S cluster in Isf is conserved in all of the homologs except two, in which either an aspartate or a histidine has replaced the second cysteine in the motif. A phylogenetic analysis of Isf homologs identified four subgroups, two of which were supported by bootstrap data. Three homologs from metabolically and phylogenetically diverse species in the Bacteria and Archaea domains (Af3 from Archaeoglobus fulgidus, Cd1 from Clostridium difficile, and Mj2 from Methanococcus jannaschii) were overproduced in Escherichia coli. Each homolog purified as a homodimer, and the UV-visible absorption spectra were nearly identical to that of Isf. After reconstitution with iron, sulfide, and flavin mononucleotide (FMN) the homologs contained six to eight nonheme iron atoms and 1.6 to 1.7 FMN molecules per dimer, suggesting that two 4Fe-4S or 3Fe-4S clusters and two FMN cofactors were bound to each dimer, which is consistent with Isf data. Homologs Af3 and Mj2 were reduced by CO in reactions catalyzed by cell extract of acetate-grown M. thermophila, but Cd1 was not. Homologs Af3 and Mj2 were reduced by CO in reactions catalyzed by A. fulgidus and M. jannaschii cell extracts. Cell extract of Clostridium thermoaceticum catalyzed CO reduction of Cd1. Our database sequence analyses and biochemical characterizations indicate that Isf is the prototype of a family of iron-sulfur flavoproteins that occur in members of all three domains.     

Design and synthesis of de novo cytochromes c

Natural c-type cytochromes are characterized by the consensus Cys-X-X-Cys-His heme-binding motif (where X is any amino acid) by which the heme is covalently attached to protein by the addition of the sulfhydryl groups of two cysteine residues to the vinyl groups of the heme. In this work, the consensus sequence was used for the heme-binding site of a designed four-helix bundle, and the apoproteins with either a histidine residue or a methionine residue positioned at the sixth coordination site were synthesized and reacted with iron protoporphyrin IX (protoheme) under mild reducing conditions in vitro. These polypeptides bound one heme per helix-loop-helix monomer via a single thioether bond and formed four-helix bundle dimers in the holo forms as designed. They exhibited visible absorption spectra characteristic of c-type cytochromes, in which the absorption bands shifted to lower wavelengths in comparison with the b-type heme binding intermediates of the same proteins. Unexpectedly, the designed cytochromes c with bis-His-coordinated heme iron exhibited oxidation-reduction potentials similar to those of their b-type intermediates, which have no thioether bond. Furthermore, the cytochrome c with His and Met residues as the axial ligands exhibited redox potentials increased by only 15-30 mV in comparison with the cytochrome with the bis-His coordination. These results indicate that highly positive redox potentials of natural cytochromes c are not only due to the heme covalent structure, including the Met ligation, but also due to noncovalent and hydrophobic environments surrounding the heme. The covalent attachment of heme to the polypeptide in natural cytochromes c may contribute to their higher redox potentials by reducing the thermodynamic stability of the oxidized forms relatively against that of the reduced forms without the loss of heme.     

Structural Characterization of Glucooligosaccharide Oxidase from Acremonium strictum

Glucooligosaccharide oxidase from Acremonium strictum was screened for potential applications in oligosaccharide acid production and carbohydrate detection. This protein is a unique covalent flavoenzyme which catalyzes the oxidation of a variety of carbohydrates with high selectivity for cello- and maltooligosaccharides. Kinetic measurements suggested that this enzyme possesses an open carbohydrate-binding groove, which is mainly composed of two glucosyl-binding subsites. The encoding gene was subsequently cloned, and one intron was detected in the genomic DNA. Large amounts of active enzymes were expressed in Pichia pastoris, with a yield of 300 mg per liter medium. The protein was predicted to share structural homology with plant cytokinin dehydrogenase and related flavoproteins that share a conserved flavin adenine dinucleotide (FAD)-binding domain. The closest sequence matches are those of plant berberine bridge enzyme-like proteins, particularly the characteristic flavinylation site. Unexpectedly, mutation of the putative FAD-attaching residue, H70, to alanine, serine, cysteine, and tyrosine did not abolish the covalent FAD linkage and had little effect on the K_m. Instead, the variants displayed k_cat values that were 50- to 600-fold lower, indicating that H70 is crucial for efficient redox catalysis, perhaps through modulation of the oxidative power of the flavin.     

Site-directed mutagenesis of the FAD-binding histidine of 6-hydroxy-D-nicotine oxidase. Consequences on flavinylation and enzyme activity

In 6-hydroxy-D-nicotine oxidase (6-HDNO) FAD is covalently bound to His71 of the polypeptide chain by an 8 alpha-(N3-histidyl)-riboflavin linkage. The FAD-binding histidine was exchanged by site-directed mutagenesis to either a Cys- or Tyr-residue, two amino acids known to be involved in covalent binding of FAD in other enzymes, or to a Ser-residue. None of the amino acid replacements for His71 allowed covalent FAD incorporation into the 6-HDNO polypeptide. Thus, the amino acid residues involved in covalent FAD-binding require a specific polypeptide surrounding in order for this modification to proceed and cannot be replaced with each other. Enzyme activity was completely abolished with Tyr in place of His71. 6-HDNO activity with non-covalently bound FAD was found with 6-HDNO-Cys and to a lesser extent also with 6-HDNO-Ser. However, the Km values for 6-HDNO-Cys and 6-HDNO-Ser were increased approximately 20-fold as compared to 6-HDNO-His. Both mutant enzymes, in contrast to the wild-type enzyme, needed additional FAD in the enzymatic assay (50 microM for 6-HDNO-Ser and 10 microM for 6-HDNO-Cys) for maximal enzyme activity.     

Characterization of the flavin association in hexose oxidase from Chondrus crispus

Hexose oxidase (EC 1.1.3.5) from Hansenula polymorpha was found to exhibit a dual covalent association of FAD with His79 via an 8 alpha-histidyl linkage as well as a covalent association between Cys138 and C-6 of the isoalloxazine moiety of FAD. Spectral properties of the wild-type enzyme exhibited maxima at 364 nm and 437 nm as well as a distinct shoulder at 445 nm. An H79K mutant enzyme exhibited only one maximum at 437 nm. The difference absorption spectrum between an oxidized and a substrate-reduced enzyme preparation showed maxima at 360 nm and 445 nm corresponding to an apparent novel type of association. Hexose oxidase showed a low, pH-independent fluorescence at 525 nm when excited at 450 nm. Flavin was released from the holoenzyme by treatment with trypsin. Sequencing of the flavopeptide revealed two peptides comprising positions 74-91 and 132-157 associated with FAD in equimolar amounts. A homology model of hexose oxidase was constructed using the crystal structure of glucooligosaccharide oxidase from Acremonium strictum as template. The model placed both of the sequences found above in the close vicinity of the FAD cofactor, and suggests covalent bonds between both His79 and Cys138 and FAD, in accordance with the chemical evidence. Based on the results, hexose oxidase is identified as incorporating FAD with a double covalent association with His79 and Cys138 in the holoenzyme. A reaction mechanism involving the concerted action of Tyr488 and Asp409 in hexose oxidase is suggested as the initiator of the proton abstraction from the substrate molecule in the active site.     

Chemical reactivity of <Emphasis Type="Italic">Synechococcus</Emphasis> sp. PCC 7002 and <Emphasis Type="Italic">Synechocystis</Emphasis> sp. PCC 6803 hemoglobins: covalent heme attachment and bishistidine coordination

In the absence of an exogenous ligand, the hemoglobins from the cyanobacteria Synechocystis sp. PCC 6803 and Synechococcus sp. PCC 7002 coordinate the heme group with two axial histidines (His46 and His70). These globins also form a covalent linkage between the heme 2-vinyl substituent and His117. The in vitro mechanism of heme attachment to His117 was examined with a combination of site-directed mutagenesis, NMR spectroscopy, and optical spectroscopy. The results supported an electrophilic addition with vinyl protonation being the rate-determining step. Replacement of His117 with a cysteine demonstrated that the reaction could occur with an alternative nucleophile. His46 (distal histidine) was implicated in the specificity of the reaction for the 2-vinyl group as well as protection of the protein from oxidative damage caused by exposure to exogenous H₂O₂.     

Evidence for a proton transfer network and a required persulfide-bond-forming cysteine residue in Ni-containing carbon monoxide dehydrogenases

Carbon monoxide dehydrogenase from Moorella thermoacetica catalyzes the reversible oxidation of CO to CO(2) at a nickel-iron-sulfur active site called the C-cluster. Mutants of a proposed proton transfer pathway and of a cysteine residue recently found to form a persulfide bond with the C-cluster were characterized. Four semiconserved histidine residues were individually mutated to alanine. His116 and His122 were essential to catalysis, while His113 and His119 attenuated catalysis but were not essential. Significant activity was "rescued" by a double mutant where His116 was replaced by Ala and His was also introduced at position 115. The activity was also rescued in double mutants where His122 was replaced by Ala and His was simultaneously introduced at either position 121 or position 123. Activity was also rescued by replacing His with Cys at position 116. Mutation of conserved Lys587 near the C-cluster attenuated activity but did not eliminate it. Activity was virtually abolished in a double mutant where Lys587 and His113 were both changed to Ala. Mutations of conserved Asn284 also attenuated activity. These effects suggest the presence of a network of amino acid residues responsible for proton transfer rather than a single linear pathway. The Ser mutant of the persulfide-forming Cys316 was essentially inactive and displayed no electron paramagnetic resonance signals originating from the C-cluster. Electronic absorption and metal analysis suggest that the C-cluster is absent in this mutant. The persulfide bond appears to be essential for either the assembly or the stability of the C-cluster, and possibly for eliciting the redox chemistry of the C-cluster required for catalytic activity.     

Molecular cloning of the gene encoding flavoredoxin, a flavoprotein from Desulfovibrio gigas

Sulfate-reducing bacteria are rich in unique redox proteins and electron carriers that participate in a variety of essential pathways. Several studies have been carried out to characterize these proteins, but the structure and function of many are poorly understood. Many Desulfovibrio species can grow using hydrogen as the sole energy source, indicating that the oxidation of hydrogen with sulfite as the terminal electron acceptor is an energy-conserving mechanism. Flavoredoxin is an FMN-binding protein isolated from the sulfate-reducing bacteria Desulfovibrio gigas that participates in the reduction of bisulfite from hydrogen. Here we report the cloning and sequencing of the flavoredoxin gene. The derived amino acid sequence exhibits similarity to several flavoproteins which are members of a new family of flavin reductases suggested to bind FMN in a novel mode.     

Covalent attachment of flavin adenine dinucleotide (FAD) and flavin mononucleotide (FMN) to enzymes: the current state of affairs.

The first identified covalent flavoprotein, a component of mammalian succinate dehydrogenase, was reported 42 years ago. Since that time, more than 20 covalent flavoenzymes have been described, each possessing one of five modes of FAD or FMN linkage to protein. Despite the early identification of covalent flavoproteins, the mechanisms of covalent bond formation and the roles of the covalent links are only recently being appreciated. The main focus of this review is, therefore, one of mechanism and function, in addition to surveying the types of linkage observed and the methods employed for their identification. Case studies are presented for a variety of covalent flavoenzymes, from which general findings are beginning to emerge.     

X-ray structure determination of a vanadium-dependent haloperoxidase from Ascophyllum nodosum at 2.0 A resolution.

The homo-dimeric structure of a vanadium-dependent haloperoxidase (V-BPO) from the brown alga Ascophyllum nodosum (EC 1.1.11.X) has been solved by single isomorphous replacement anomalous scattering (SIRAS) X-ray crystallography at 2.0 A resolution (PDB accession code 1QI9), using two heavy-atom datasets of a tungstate derivative measured at two different wavelengths. The protein sequence (SwissProt entry code P81701) of V-BPO was established by combining results from protein and DNA sequencing, and electron density interpretation. The enzyme has nearly an all-helical structure, with two four-helix bundles and only three small beta-sheets. The holoenzyme contains trigonal-bipyramidal coordinated vanadium atoms at its two active centres. Structural similarity to the only other structurally characterized vanadium-dependent chloroperoxidase (V-CPO) from Curvularia inaequalis exists in the vicinity of the active site and to a lesser extent in the central four-helix bundle. Despite the low sequence and structural similarity between V-BPO and V-CPO, the vanadium binding centres are highly conserved on the N-terminal side of an alpha-helix and include the proposed catalytic histidine residue (His418(V-BPO)/His404(V-CPO)). The V-BPO structure contains, in addition, a second histidine near the active site (His411(V-BPO)), which can alter the redox potential of the catalytically active VO2-O2 species by protonation/deprotonation reactions. Specific binding sites for the organic substrates, like indoles and monochlordimedone, or for halide ions are not visible in the V-BPO structure. A reaction mechanism for the enzymatic oxidation of halides is discussed, based on the present structural, spectroscopic and biochemical knowledge of vanadium-dependent haloperoxidases, explaining the observed enzymatic differences between both enzymes.     

Mercuric reductase. Purification and characterization of a transposon-encoded flavoprotein containing an oxidation-reduction-active disulfide

The flavoprotein mercuric reductase catalyzes the two-electron reduction of mercuric ions to elemental mercury using NADPH as an electron donor. It has now been purified from Pseudomonas aeruginosa PAO9501 carrying the plasmid pVS1. In this plasmid system, where the mer operon is on the transposon Tn501, mercuric reductase comprises up to 6% of the soluble cellular protein upon induction with mercurials. The purification is a rapid (two-step), high yield (80%) procedure. Anaerobic titrations of mercuric reductase with dithionite revealed the formation of a charge transfer complex with an absorbance maximum around 540 nm. Striking spectroscopic similarities to lipoamide dehydrogenase and glutathione reductase were observed. These two enzymes, which catalyze the transfer of electrons between pyridine nucleotides and disulfides, are flavoproteins which contain an oxidation-reduction-active cysteine residue at the active site. The expectation that mercuric reductase contains a similar electron acceptor was confirmed when it was shown that mercuric reductase has the capacity to accept four electrons per FAD-containing subunit, and that two thiols become kinetically titrable by 5,5'-dithiobis-(2-nitrobenzoate) upon reduction with NADPH. These are characteristic features of the disulfide reductase class of flavoproteins. Further similarities with at least one of these enzymes, lipoamide dehydrogenase, include the E/EH2 midpoint potential (-269 mV), fluorescence properties, and extinction coefficients of E and EH2. Preliminary observations relevant to an understanding of the mechanism of mercuric reductase are discussed.