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MtsZ is a molybdenum-containing methionine sulfoxide reductase that supports virulence in the human respiratory pathogen Haemophilus influenzae (Hi). HiMtsZ belongs to a group of structurally and spectroscopically uncharacterized S-/N-oxide reductases, all of which are found in bacterial pathogens. Here, we have solved the crystal structure of HiMtsZ, which reveals that the HiMtsZ substrate-binding site encompasses a previously unrecognized part that accommodates the methionine sulfoxide side chain via interaction with His182 and Arg166. Charge and amino acid composition of this side chain-binding region vary and, as indicated by electrochemical, kinetic, and docking studies, could explain the diverse substrate specificity seen in closely related enzymes of this type. The HiMtsZ Mo active site has an underlying structural flexibility, where dissociation of the central Ser187 ligand affected catalysis at low pH. Unexpectedly, the two main HiMtsZ electron paramagnetic resonance (EPR) species resembled not only a related dimethyl sulfoxide reductase but also a structurally unrelated nitrate reductase that possesses an Asp-Mo ligand. This suggests that contrary to current views, the geometry of the Mo center and its primary ligands, rather than the specific amino acid environment, is the main determinant of the EPR properties of mononuclear Mo enzymes. The flexibility in the electronic structure of the Mo centers is also apparent in two of three HiMtsZ EPR-active Mo(V) species being catalytically incompetent off-pathway forms that could not be fully oxidized., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Zinc (Zn) and copper (Cu) are essential for optimal innate immune function, and nutritional deficiency in either metal leads to increased susceptibility to bacterial infection. Recently, the decreased survival of bacterial pathogens with impaired Cu and/or Zn detoxification systems in phagocytes and animal models of infection has been reported. Consequently, a model has emerged in which the host utilizes Cu and/or Zn intoxication to reduce the intracellular survival of pathogens. This review describes and assesses the potential role for Cu and Zn intoxication in innate immune function and their direct bactericidal function., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

In prototypic Escherichia coli K-12 the introduction of disulfide bonds into folding proteins is mediated by the Dsb family of enzymes, primarily through the actions of the highly oxidizing protein EcDsbA. Homologues of the Dsb catalysts are found in most bacteria. Interestingly, pathogens have developed distinct Dsb machineries that play a pivotal role in the biogenesis of virulence factors, hence contributing to their pathogenicity. Salmonella enterica serovar (sv.) Typhimurium encodes an extended number of sulfhydryl oxidases, namely SeDsbA, SeDsbL, and SeSrgA. Here we report a comprehensive analysis of the sv. Typhimurium thiol oxidative system through the structural and functional characterization of the three Salmonella DsbA paralogues. The three proteins share low sequence identity, which results in several unique three-dimensional characteristics, principally in areas involved in substrate binding and disulfide catalysis. Furthermore, the Salmonella DsbA-like proteins also have different redox properties. Whereas functional characterization revealed some degree of redundancy, the properties of SeDsbA, SeDsbL, and SeSrgA and their expression pattern in sv. Typhimurium indicate a diverse role for these enzymes in virulence.

Mutants of the active site residues Trp-116 and Tyr-114 of the molybdenum-containing Me(2)SO reductase from Rhodobacter capsulatus have been examined spectroscopically and kinetically. The Y114F mutant has an increased rate constant for oxygen atom transfer from Me(2)SO to reduced enzyme, the result of lower stability of the E(red).Me(2)SO complex. The absorption spectrum of this species (but not that of either oxidized or reduced enzyme) is significantly perturbed in the mutant relative to wild-type enzyme, consistent with Tyr-114 interacting with bound Me(2)SO. The as-isolated W116F mutant is only five-coordinate, with one of the two equivalents of the pyranopterin cofactor found in the enzyme dissociated from the molybdenum and replaced by a second Mo=O group. Reduction of the mutant with sodium dithionite and reoxidation with Me(2)SO, however, regenerates the long-wavelength absorbance of functional enzyme, although the wavelength maximum is shifted to 670 nm from the 720 nm of wild-type enzyme. This "redox-cycled" mutant exhibits a Me(2)SO reducing activity and overall reaction mechanism similar to that of wild-type enzyme but rapidly reverts to the inactive five-coordinate form in the course of turnover.

Cytochromes from the SoxAX family have a major role in thiosulfate oxidation via the thiosulfate-oxidizing multi-enzyme system (TOMES). Previously characterized SoxAX proteins from Rhodovulum sulfidophilum and Paracoccus pantotrophus contain three heme c groups, two of which are located on the SoxA subunit. In contrast, the SoxAX protein purified from Starkeya novella was found to contain only two heme groups. Mass spectrometry showed that a disulfide bond replaced the second heme group found in the diheme SoxA subunits. Apparent molecular masses of 27,229 +/- 10.3 Da and 20,258.6 +/- 1 Da were determined for SoxA and SoxX with an overall mass of 49.7 kDa, indicating a heterodimeric structure. Optical redox potentiometry found that the two heme cofactors are reduced at similar potentials (versus NHE) that are as follows: +133 mV (pH 6.0); +104 mV (pH 7.0); +49 (pH 7.9) and +10 mV (pH 8.7). EPR spectroscopy revealed that both ferric heme groups are in the low spin state, and the spectra were consistent with one heme having a His/Cys axial ligation and the other having a His/Met axial ligation. The His/Cys ligated heme is present in different conformational states and gives rise to three distinct signals. Amino acid sequencing was used to unambiguously assign the protein to the encoding genes, soxAX, which are part of a complete sox gene cluster found in S. novella. Phylogenetic analysis of soxA- and soxX-related gene sequences indicates a parallel development of SoxA and SoxX, with the diheme and monoheme SoxA sequences located on clearly separated branches of a phylogenetic tree.

Direct oxidation of sulfite to sulfate occurs in various photo- and chemotrophic sulfur oxidizing microorganisms as the final step in the oxidation of reduced sulfur compounds and is catalyzed by sulfite:cytochrome c oxidoreductase (EC ). Here we show that the enzyme from Thiobacillus novellus is a periplasmically located alphabeta heterodimer, consisting of a 40.6-kDa subunit containing a molybdenum cofactor and an 8.8-kDa mono-heme cytochrome c(552) subunit (midpoint redox potential, E(m8.0) = +280 mV). The organic component of the molybdenum cofactor was identified as molybdopterin contained in a 1:1 ratio to the Mo content of the enzyme. Electron paramagnetic resonance spectroscopy revealed the presence of a sulfite-inducible Mo(V) signal characteristic of sulfite:acceptor oxidoreductases. However, pH-dependent changes in the electron paramagnetic resonance signal were not detected. Kinetic studies showed that the enzyme exhibits a ping-pong mechanism involving two reactive sites. K(m) values for sulfite and cytochrome c(550) were determined to be 27 and 4 micrometer, respectively; the enzyme was found to be reversibly inhibited by sulfate and various buffer ions. The sorAB genes, which encode the enzyme, appear to form an operon, which is preceded by a putative extracytoplasmic function-type promoter and contains a hairpin loop termination structure downstream of sorB. While SorA exhibits significant similarities to known sequences of eukaryotic and bacterial sulfite:acceptor oxidoreductases, SorB does not appear to be closely related to any known c-type cytochromes.

The dorC gene of the dimethyl sulfoxide respiratory (dor) operon of Rhodobacter capsulatus encodes a pentaheme c-type cytochrome that is involved in electron transfer from ubiquinol to periplasmic dimethyl sulfoxide reductase. DorC was expressed as a C-terminal fusion to an 8-amino acid FLAG epitope and was purified from detergent-solubilized membranes by ion exchange chromatography and immunoaffinity chromatography. The DorC protein had a subunit Mr = 46,000, and pyridine hemochrome analysis indicated that it contained 5 mol heme c/mol DorC polypeptide, as predicted from the derived amino acid sequence of the dorC gene. The reduced form of DorC exhibited visible absorption maxima at 551.5 nm (alpha-band), 522 nm (beta-band), and 419 nm (Soret band). Redox potentiometry of the heme centers of DorC identified five components (n = 1) with midpoint potentials of -34, -128, -184, -185, and -276 mV. Despite the low redox potentials of the heme centers, DorC was reduced by duroquinol and was oxidized by dimethyl sulfoxide reductase.