The Roles of Cyma in Support of the Respiratory Flexibility of Shewanella

Electron Transfer at the Microbe–Mineral Interface 1217 The roles of CymA in support of the respiratory flexibility of Shewanella oneidensis MR-1 Sophie J. Marritt*, Duncan G.G. McMillan†, Liang Shi‡, James K. Fredrickson‡, John M. Zachara‡, David J. Richardson*, Lars J.C. Jeuken† and Julea N. Butt*1 *Centre for Molecular and Structural Biochemistry, School of Chemistry and School of Biological Sciences, University of East Anglia, Norwich Research Park, Norwich NR4 7TJ, U.K., †Institute of Membrane and Systems Biology, Centre for Molecular Nanoscience, School of Physics and Astronomy, University of Leeds, Woodhouse Lane, Leeds LS2 9JT, U.K., and ‡Pacific Northwest National Laboratory, 902 Battelle Boulevard, Richland, WA 99352, U.S.A. Abstract Shewanella species are isolated from the oxic/anoxic regions of seawater and aquatic sediments where redox conditions fluctuate in time and space. Colonization of these environments is by virtue of flexible respiratory chains, many of which are notable for the ability to reduce extracellular substrates including the Fe(III) and Mn(IV) contained in oxide and phyllosilicate minerals. Shewanella oneidensis MR-1 serves as a model organism to consider the biochemical basis of this flexibility. In the present paper, we summarize the various systems that serve to branch the respiratory chain of S. oneidensis MR-1 in order that electrons from quinol oxidation can be delivered the various terminal electron acceptors able to support aerobic and anaerobic growth. This serves to highlight several unanswered questions relating to the regulation of respiratory electron transport in Shewanella and the central role(s) of the tetrahaem-containing quinol dehydrogenase CymA in that process. Introduction is generated per QH2 molecule oxidized can be varied to meet The gammaproteobacterium Shewanella oneidensis MR-1 physiological need. colonizes various changeable marine and freshwater environ- Three terminal oxidases that catalyse the respiratory ments. This capability is underpinned by a flexible respiratory reduction of O2 have been identified in S. oneidensis MR- system able to couple the oxidation of molecules such as 1. Cytochrome bd contributes to the pmf without being lactate, pyruvate, protein and DNA to the reduction of a range a proton pump by virtue of its ability to oxidize QH2 of terminal electron acceptors located either within or outside and deliver the two released protons to the periplasm. It the cell (Table 1). These electron-transfer events release free is likely to operate as a high-affinity oxidase at low O2 energy that can be harnessed in the form of a pmf (proton- tensions or under growth conditions that may be considered motive force) across the microbial inner membrane. The pmf stressful to S. oneidensis MR-1. The cytochrome aa3-and has both a chemical (pH) and an electrical (ψ) component, cbb3-type oxidases are proton pumps likely to operate under and it is directly responsible for driving ATP synthesis. O2-replete and -depleted conditions respectively. These Whichever combination of respiratory electron donor and terminal oxidases receive electrons from the action of the acceptor contribute to maintaining the pmf, a key component QH2-oxidizing cytochrome bc1 complex via cytochrome c. of the electron-transfer chain is the redox cycling of Qs Cytochrome bc1 contributes to maintaining the pmf through (quinones) [1]. Qs are diffusible lipophilic molecules that are the Q-cycle, so six protons are translocated across the inner confined to the microbial inner membrane where they membrane per QH2 oxidized when the cytochrome aa3-and are reduced to QH2s (quinols) by the reversible addition cbb3-type enzymes reduce O2 [3]. of two electrons and two protons. During aerobic and microaerobic growth, O2 serves as terminal electron acceptor with the immediate electron donor to the Q-pool CymA: a constitutive QH2 dehydrogenase being respectively NADH, via the action of NADH:Q Another QH2 dehydrogenase present during aerobic and oxidoreductases, and formate, through the action of formate microaerobic growth of S. oneidensis MR-1 is CymA [4]. dehydrogenases [2]. Several enzymes then allow QH2 This enzyme is a member of the NapC/NirT family of oxidation to be coupled to O2 reduction (Table 1). The result QH2 dehydrogenases. It possesses a single-transmembrane is a branched electron-transport chain in which the pmf that α-helix and a periplasmic globular domain containing four c-type haems. Magnetic circular dichroism has established that three of these haems have histidine–histidine axial lig- Key words: cytochrome, electrochemistry, haem, mineral reduction, quinone, Shewanella ation [5]. The fourth haem has histidine–water axial oneidensis. ligation and forms an intrinsic part of the QH2-oxidation Abbreviations used: MMQ, methylmenaquinone; MQ, menaquinone; pmf, protonmotive force; Q, quinone; QH , quinol; UQ, ubiquinone; UQH , ubiquinol. site. Spectropotentiometric titration has defined midpoint 2 2 ◦ 1 To whom correspondence should be addressed (email [email protected]). potentials (E ) at pH 7 of approximately − 110, − 190 and C C Biochemical Society Transactions www.biochemsoctrans.org Biochem. Soc. Trans. (2012) 40, 1217–1221; doi:10.1042/BST20120150 The Authors Journal compilation 2012 Biochemical Society 1218 Biochemical Society Transactions (2012) Volume 40, part 6 Table 1 Summary of terminal reductase systems that can operate in S. oneidensis MR-1 Site of terminal electron Quinol Terminal electron acceptor Terminal reductase acceptor reduction dehydrogenase Reference(s) O2 Cytochrome c oxidase (SO4606–SO4609) Inner membrane Cytochrome bc1 complex (SO0608–SO0610) Cytochrome cbb3 (SO2361–SO2364) Inner membrane Cytochrome bc1 complex Cytochrome bd (SO3285–SO3286) Inner membrane Intrinsic H2O2?CcpA(SO2178) Periplasm CymA? [7] − NO3 NapAB (SO0845, SO0848) Periplasm NapGH (SO0846, [11] SO0847)CymA (SO4591) − NO2 NrfA (SO3980) Periplasm CymA [11] Otr (SO4144) Periplasm CymA? [23] Fumarate Fcc3 (SO0970) Periplasm CymA [8,10] Ifc3 (SO1421) Periplasm CymA [8] FrdABC (SO0396–SO0399) Cytoplasmic face of inner Intrinsic (FrdC) membrane Trimethylamine N-oxide TorA (SO1232) Periplasm TorC (SO1233) [24,25] DMSO DMSO reductase, dmsAB-1 (SO1427–SO1430) Extracellular CymA [26] DMSO reductase, dmsAB-2 (SO4357–SO4360) Extracellular CymA Insoluble Fe(III) MtrCAB, OmcA, MtrDEF (SO1776–SO1782) Extracellular CymA [27] Soluble Fe(III) ??? Periplasm CymA [20,28,29] 2 − S4O6 Otr (SO4144) Periplasm CymA? [23] Sulfur Polysulfide reductase, PsrABC (SO4060–SO4062) Periplasm Intrinsic (PsrC) [30] 2 − 2 − SO3 ,S2O3 Octahaem sulfite reductase, SirA (SO0479) Periplasm SirCD (SO0483, [31] SO0484) − 265 mV for the three low-spin haems and approximately and perhaps allow peroxide to serve as a terminal electron − 240 mV for the high-spin haem; all potentials are quoted acceptor. compared with the SHE (standard hydrogen electrode). cymA is expressed constitutively, but expression can These E◦ values are consistent with the envelopes of reductive increase during anaerobic growth where, in contrast with the and oxidative current visualized by cyclic voltammetry of situation in aerobic growth, the physiological contributions CymA adsorbed on gold electrodes [6]. Similar behaviour is of CymA are well established. CymA serves as a QH2 seen for CymA adsorbed on graphite electrodes (Figure 1). dehydrogenase that is able to supply electrons to numerous To investigate whether CymA reacts with O2, an aliquot of protein and enzyme systems dedicated to reducing a variety air-equilibrated buffer was introduced into the voltammetric of terminal electron acceptors (Table 1). Deletion of cymA experiment (Figure 1). This resulted in the appearance of a has shown that the gene product is essential for the terminal clear catalytic reduction wave where the onset of catalysis reduction of fumarate, nitrate, nitrite, soluble complexes of at approximately − 100 mV coincides with reduction of Fe(III) and extracellular substrates such as minerals of Fe(III) the haem cofactors. The catalytic currents are also observed and Mn(IV), particulate DMSO and electrode materials [8– at potentials that are significantly higher than required to 12]. CymA may also provide electrons to the periplasmic observe the direct reduction of O2 by the electrode at a octahaem tetrathionate reductase for which genomic analysis comparable rate (Figure 1). Thus CymA can reduce O2. fails to find evidence for a dedicated QH2 dehydrogenase. Whether this reaction occurs in vivo and, if it does, the CymA is not in itself protonmotive. However, it may nature of its contribution to the physiological demands contribute to pmf generation as part of a redox loop since of the cell remain to be established. In vitro experiments QH2 oxidation releases two electrons and two protons into have demonstrated electron transfer from CymA to the the periplasm. When formate feeds electrons to the Q- cytochrome c peroxidase CcpA via monohaem cytochrome pool via FdhABC and the electrons are vented via CymA, ScyA [7]. CcpA removes potentially harmful peroxides and pmf generation would appear to be solely at the level of hydroxide radicals produced in the periplasm as unwanted electron input into the Q-pool. However, S. oneidensis MR- products of O2 reduction. Electron transfer from CymA 1 was reported to translocate 0.14 and 0.47 protons per to CcpA may afford S. oneidensis MR-1 protection against two electrons during the CymA-dependent reduction of reactive oxygen species produced in fluctuating oxygen

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