Quinol-cytochrome c Oxidoreductase and Cytochrome c4 Mediate Electron Transfer during Selenate Respiration in Thauera selenatis

Elisabeth C Lowe,Sarah Bydder,Robert S Hartshorne,Hannah L.U Tape,Elizabeth J Dridge,Charles M Debieux,Konrad Paszkiewicz,Ian Singleton,Richard J Lewis,Joanne M Santini,David J Richardson,Clive S Butler

doi:10.1074/jbc.m110.115873

Abstract

Selenate reductase (SER) from Thauera selenatis is a periplasmic enzyme that has been classified as a type II molybdoenzyme. The enzyme comprises three subunits SerABC, where SerC is an unusual b-heme cytochrome. In the present work the spectropotentiometric characterization of the SerC component and the identification of redox partners to SER are reported. The mid-point redox potential of the b-heme was determined by optical titration (E(m) + 234 +/- 10 mV). A profile of periplasmic c-type cytochromes expressed in T. selenatis under selenate respiring conditions was undertaken. Two c-type cytochromes were purified ( approximately 24 and approximately 6 kDa), and the 24-kDa protein (cytc-Ts4) was shown to donate electrons to SerABC in vitro. Protein sequence of cytc-Ts4 was obtained by N-terminal sequencing and liquid chromatography-tandem mass spectrometry analysis, and based upon sequence similarities, was assigned as a member of cytochrome c(4) family. Redox potentiometry, combined with UV-visible spectroscopy, showed that cytc-Ts4 is a diheme cytochrome with a redox potential of +282 +/- 10 mV, and both hemes are predicted to have His-Met ligation. To identify the membrane-bound electron donors to cytc-Ts4, growth of T. selenatis in the presence of respiratory inhibitors was monitored. The specific quinol-cytochrome c oxidoreductase (QCR) inhibitors myxothiazol and antimycin A partially inhibited selenate respiration, demonstrating that some electron flux is via the QCR. Electron transfer via a QCR and a diheme cytochrome c(4) is a novel route for a member of the DMSO reductase family of molybdoenzymes.

Highlights

Selenate reductase (SER) from Thauera selenatis is a periplasmic enzyme that has been classified as a type II molybdoenzyme
To test the ability of this cytochrome to donate electrons to SerABC, it was reduced by titration with a weak solution of sodium dithionite, and mixed with purified selenate reductase and selenate in an anaerobic assay
No re-oxidation of the heme was observed suggesting that the 6-kDa cytochrome is not able to act as an electron donor to SerABC in vitro

Summary

Introduction

Selenate reductase (SER) from Thauera selenatis is a periplasmic enzyme that has been classified as a type II molybdoenzyme. Within the DMSO reductase family of type II molybdoenzymes [1] there is a distinct clade of enzymes that are translocated to the periplasm using the twin arginine translocation (TAT) pathway [2, 3] and possess a monomeric b-type heme-containing ␥-subunit [1] The enzymes within this clade function as either dehydrogenases (e.g. ethylbenzene dehydrogenase (EBDH) from Aromatoleum aromaticum [4] and dimethylsulfide dehydrogenase from Rhodovulum sulfidophilum [1, 5]) or reductases (e.g. selenate reductase from Thauera selenatis [6, 7] and chlorate reductase from Ideonella dechloratans [8, 9]) and catalyze either hydride or oxygen transfer as generalized by Reaction 1. Analysis of the amino acid sequence alignment of EBDH and DMSDH with selenate reductase (SER) shows that the coordinating methionine (Met-138) and lysine (Lys-228) residues are conserved in SerC This raises interesting questions as to what is the redox potential for the b-heme in the reductase members of this distinct type II clade and what are the likely redox partners?

Objectives

Results

Conclusion