Sulfide-Quinone Reductase from Rhodobacter capsulatus: PURIFICATION, CLONING, AND EXPRESSION

Michael Schütz,Yosepha Shahak,Etana Padan,Günter Hauska

doi:10.1074/jbc.272.15.9890

Abstract

A sulfide-quinone oxidoreductase (SQR, EC 1.8.5.'.) has been purified to homogeneity from chromatophores of the non-sulfur purple bacterium Rhodobacter capsulatus DSM 155. It is composed of a single polypeptide with an apparent molecular mass of about 55 kDa, exhibiting absorption and fluorescence spectra typical for a flavoprotein and similar to the SQR from the cyanobacterium Oscillatoria limnetica. From N-terminal and tryptic peptide sequences of the pure protein a genomic DNA clone was obtained by polymerase chain reaction amplification. Its sequence contains an open reading frame of 1275 base pairs (EMBL nucleotide sequence data base, accession no. X97478X97478) encoding the SQR of R. capsulatus. The deduced polypeptide consists of 425 amino acid residues with a molecular mass of 47 kDa and a net charge of +9. The high similarity (72%)/identity (48%) between the N termini of the cyanobacterial and the bacterial enzyme was confirmed and extended. Both enzymes exhibit the FAD/NAD(P) binding betaalphabeta-fold (Wierenga, R. K., Terpstra, P., and Hol, W. G. S. (1986) J. Mol. Biol. 187, 101-107). The complete sequence of the SQR from R. capsulatus shows further similarity to flavoproteins, in particular glutathione reductase and lipoamide dehydrogenase. The cloned sqr was expressed in Escherichia coli in a functional form.

Highlights

A sulfide-quinone oxidoreductase (SQR, EC 1.8.5.؅.) has been purified to homogeneity from chromatophores of the non-sulfur purple bacterium Rhodobacter capsulatus DSM 155. It is composed of a single polypeptide with an apparent molecular mass of about 55 kDa, exhibiting absorption and fluorescence spectra typical for a flavoprotein and similar to the sulfidequinone reductase (SQR) from the cyanobacterium Oscillatoria limnetica
In cyanobacteria this property has been demonstrated and most extensively studied in Oscillatoria limnetica, which is capable of both oxygenic and sulfide-oxidizing, anoxygenic photosynthesis [1]. Anoxygenic photosynthesis in this organism is induced by sulfide, and the inducible factor was characterized as a sulfidequinone reductase (SQR)1 by Arieli et al [2]
In the present work we describe the purification to homogeneity and the characterization of SQR from the purple photosynthetic bacterium R. capsulatus DSM 155

Summary

Introduction

A sulfide-quinone oxidoreductase (SQR, EC 1.8.5.؅.) has been purified to homogeneity from chromatophores of the non-sulfur purple bacterium Rhodobacter capsulatus DSM 155. From N-terminal and tryptic peptide sequences of the pure protein a genomic DNA clone was obtained by polymerase chain reaction amplification. The complete sequence of the SQR from R. capsulatus shows further similarity to flavoproteins, in particular glutathione reductase and lipoamide dehydrogenase.

Results

Conclusion