Item

Abstract

Many Bacteria and Archaea employ a novel pathway of sulfur oxidation involving an enzyme complex that is related to the heterodisulfide reductase (HdrABC) of methanogens. As a first step in the biochemical characterization of Hdr‐like proteins from sulfur oxidizers (sHdr), we structurally analyzed the recombinant sHdrA protein from the Alphaproteobacterium Hyphomicrobium denitrificans at 1.4 Å resolution. The sHdrA core structure is similar to that of methanogenic HdrA (mHdrA) which binds the electron‐bifurcating flavin adenine dinucleotide (FAD), the heart of the HdrABC‐[NiFe]‐hydrogenase catalyzed reaction. Each sHdrA homodimer carries two FADs and two [4Fe‐4S] clusters being linked by electron conductivity. Redox titrations monitored by electron paramagnetic resonance and visible spectroscopy revealed a redox potential between ‐203 and ‐188 mV for the [4Fe‐4S] center. The potentials for the FADH•/FADH– and FAD/FADH• pairs reside between ‐174 and ‐156 mV and between ‐81 and ‐19 mV, respectively. The resulting stable semiquinone FADH• species, already detectable in the visible and EPR spectra of the as‐isolated state of sHdrA is incompatible with basic principles of flavin‐based electron bifurcation such that the sHdr complex does not apply this new mode of energy coupling. The inverted one‐electron FAD redox potentials of sHdr and mHdr are clearly reflected in the different FAD‐polypeptide interactions. According to this finding and the assumption that the sHdr complex forms an asymmetric HdrAA’B1C1B2C2 hexamer we tentatively propose a mechanism that links protein‐bound sulfane oxidation to sulfite on HdrB1 with NAD⁺ reduction via lipoamide disulfide reduction on HdrB2. The FAD of HdrA thereby serves as an electron storage unit.