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Staphylococcus aureus sqr Encodes a Type II Sulfide:Quinone Oxidoreductase and Impacts Reactive Sulfur Speciation in Cells.

ABSTRACT: Recent studies implicate hydrogen sulfide (H2S) oxidation as an important aspect of bacterial antibiotic resistance and sulfide homeostasis. The cst operon of the major human pathogen Staphylococcus aureus is induced by exogenous H2S stress and encodes enzymes involved in sulfide oxidation, including a group I flavoprotein disulfide oxidoreductase sulfide:quinone oxidoreductase (SQR). In this work, we show that S. aureus SQR catalyzes the two-electron oxidation of sodium sulfide (Na2S) into sulfane sulfur (S0) when provided flavin adenine dinucleotide and a water-soluble quinone acceptor. Cyanide, sulfite, and coenzyme A (CoA) are all capable of functioning as the S0 acceptor in vitro. This activity requires a C167-C344 disulfide bond in the resting enzyme, with the intermediacy of a C344 persulfide in the catalytic cycle, verified by mass spectrometry of sulfide-reacted SQR. Incubation of purified SQR and S. aureus CstB, a known FeII persulfide dioxygenase-sulfurtransferase also encoded by the cst operon, yields thiosulfate from sulfide, in a CoA-dependent manner, thus confirming the intermediacy of CoASSH as a product and substrate of SQR and CstB, respectively. Sulfur metabolite profiling of wild-type, ?sqr, and ?sqr::pSQR strains reveals a marked and specific elevation in endogenous levels of CoASSH and inorganic tetrasulfide in the ?sqr strain. We conclude that SQR impacts the cellular speciation of these reactive sulfur species but implicates other mechanisms not dependent on SQR in the formation of low-molecular weight thiol persulfides and inorganic polysulfides during misregulation of sulfide homeostasis.

SUBMITTER: Shen J

PROVIDER: S-EPMC5423654 | biostudies-literature | 2016 Nov

REPOSITORIES: biostudies-literature

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Staphylococcus aureus sqr Encodes a Type II Sulfide:Quinone Oxidoreductase and Impacts Reactive Sulfur Speciation in Cells.

Shen Jiangchuan J Peng Hui H Zhang Yixiang Y Trinidad Jonathan C JC Giedroc David P DP

Biochemistry 20161116 47

Recent studies implicate hydrogen sulfide (H<sub>2</sub>S) oxidation as an important aspect of bacterial antibiotic resistance and sulfide homeostasis. The cst operon of the major human pathogen Staphylococcus aureus is induced by exogenous H<sub>2</sub>S stress and encodes enzymes involved in sulfide oxidation, including a group I flavoprotein disulfide oxidoreductase sulfide:quinone oxidoreductase (SQR). In this work, we show that S. aureus SQR catalyzes the two-electron oxidation of sodium su ...[more]

PMID: 27806570

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Project description:Monotopic membrane-bound flavoproteins, sulfide:quinone oxidoreductases (SQRs), have a variety of physiological functions, including sulfide detoxification. SQR enzymes are classified into six groups. SQRs use the flavin adenine dinucleotide (FAD) cofactor to transfer electrons from sulfide to quinone. A type VI SQR of the photosynthetic purple sulfur bacterium, Thiocapsa roseopersicina (TrSqrF), has been previously characterized, and the mechanism of sulfide oxidation has been proposed. This paper reports the characterization of quinone binding site (QBS) of TrSqrF composed of conserved aromatic and apolar amino acids. Val331, Ile333, and Phe366 were identified near the benzoquinone ring of enzyme-bound decylubiquinone (dUQ) using the TrSqrF homology model. In silico analysis revealed that Val331 and Ile333 alternately connected with the quinone head group via hydrogen bonds, and Phe366 and Trp369 bound the quinones via hydrophobic interactions. TrSqrF variants containing alanine (V331A, I333A, F366A) and aromatic amino acid (V331F, I333F, F366Y), as well as a C-terminal α-helix deletion (CTD) mutant were generated. These amino acids are critical for quinone binding and, thus, catalysis. Spectroscopic analyses proved that all mutants contained FAD. I333F replacement resulted in the lack of the charge transfer complex. In summary, the interactions described above maintain the quinone molecule's head in an optimal position for direct electron transfer from FAD. Surprisingly, the CTD mutant retained a relatively high level of specific activity while remaining membrane-anchored. This is a unique study because it focuses on the QBS and the oxidative stage of a type VI sulfide-dependent quinone reduction. KEY POINTS: • V331, I333, F366, and W369 were shown to interact with decylubiquinone in T. roseopersicina SqrF • These amino acids are involved in proper positioning of quinones next to FAD • I333 is essential in formation of a charge transfer complex from FAD to quinone.

Project description:Staphylococcus aureus is a commensal human pathogen and a major cause of nosocomial infections. As gaseous signaling molecules, endogenous hydrogen sulfide (H2S) and nitric oxide (NO·) protect S. aureus from antibiotic stress synergistically, which we propose involves the intermediacy of nitroxyl (HNO). Here, we examine the effect of exogenous sulfide and HNO on the transcriptome and the formation of low-molecular-weight (LMW) thiol persulfides of bacillithiol, cysteine, and coenzyme A as representative of reactive sulfur species (RSS) in wild-type and ?cstR strains of S. aureus. CstR is a per- and polysulfide sensor that controls the expression of a sulfide oxidation and detoxification system. As anticipated, exogenous sulfide induces the cst operon but also indirectly represses much of the CymR regulon which controls cysteine metabolism. A zinc limitation response is also observed, linking sulfide homeostasis to zinc bioavailability. Cellular RSS levels impact the expression of a number of virulence factors, including the exotoxins, particularly apparent in the ?cstR strain. HNO, like sulfide, induces the cst operon as well as other genes regulated by exogenous sulfide, a finding that is traced to a direct reaction of CstR with HNO and to an endogenous perturbation in cellular RSS, possibly originating from disassembly of Fe-S clusters. More broadly, HNO induces a transcriptomic response to Fe overload, Cu toxicity, and reactive oxygen species and reactive nitrogen species and shares similarity with the sigB regulon. This work reveals an H2S/NO· interplay in S. aureus that impacts transition metal homeostasis and virulence gene expression. IMPORTANCE Hydrogen sulfide (H2S) is a toxic molecule and a recently described gasotransmitter in vertebrates whose function in bacteria is not well understood. In this work, we describe the transcriptomic response of the major human pathogen Staphylococcus aureus to quantified changes in levels of cellular organic reactive sulfur species, which are effector molecules involved in H2S signaling. We show that nitroxyl (HNO), a recently described signaling intermediate proposed to originate from the interplay of H2S and nitric oxide, also induces changes in cellular sulfur speciation and transition metal homeostasis, thus linking sulfide homeostasis to an adaptive response to antimicrobial reactive nitrogen species.

Dataset Information

Staphylococcus aureus sqr Encodes a Type II Sulfide:Quinone Oxidoreductase and Impacts Reactive Sulfur Speciation in Cells.

Publications

Staphylococcus aureus sqr Encodes a Type II Sulfide:Quinone Oxidoreductase and Impacts Reactive Sulfur Speciation in Cells.

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