Novel [2Fe-2S]-type Redox Center C in SdhC of Archaeal Respiratory Complex II from Sulfolobus tokodaii Strain 7

Toshio Iwasaki,Asako Kounosu,Miho Aoshima,Daijiro Ohmori,Takeo Imai,Akio Urushiyama,Nathaniel J Cosper,Robert A Scott

doi:10.1074/jbc.m207312200

Abstract

The SdhC subunit of the archaeal respiratory complex II (succinate:quinone oxidoreductase) from Sulfolobus tokodaii strain 7 has a novel cysteine rich motif and is also related to archaeal and bacterial heterodisulfide reductase subunits. We overexpressed the sdhC gene heterologously in Escherichia coli and characterized the gene product in greater detail. Low temperature resonance Raman and x-ray absorption spectroscopic investigation collectively demonstrate the presence of a [2Fe-2S] cluster core with complete cysteinyl ligation (Center C) and an isolated zinc site in the recombinant SdhC. The [2Fe-2S]2+ cluster core is sensitive to dithionite, resulting in irreversible breakdown of the Fe-Fe interaction. EPR analysis confirmed that the novel Center C is an inherent redox center in the archaeal complex II, showing unique EPR signals in the succinate-reduced state. Distinct subunit and cofactor arrangements in the S. tokodaii respiratory complex II, as compared with those in mitochondrial and some mesophilic bacterial enzymes, indicate modular evolution of this ubiquitous electron entry site in the respiratory chains of aerobic organisms.

Highlights

Respiratory complex II is an iron-sulfur (FeS)1 flavoprotein complex that serves as the sole membrane-bound component of the oxidative tricarboxylic acid cycle as well as one of the most important primary dehydrogenases at the electron entry site of the aerobic respiratory chain for a variety of aerobic organisms from archaea to bacteria to eukarya [1,2,3,4]
Marked variation was noted between the membrane anchor subunits involved in the electron transfer reactions, which bind two menaquinone molecules in the E. coli complex [5] and two protoheme centers in the W. succinogenes complex [6], both arranged vertically relative to the cytoplasmic membranes
Based on the reported crystal structures of bacterial fumarate reductase (Frd) complexes [5, 6], these results suggest that the S. tokodaii SdhAB subcomplex has a similar nearly linear cofactor arrangement with the sequence FAD-[2Fe-2S]-[4Fe-4S]-[4Fe-4S] [13]

Summary

EXPERIMENTAL PROCEDURES

E. coli strain DH5␣ and strain HB101, which were purchased from TaKaRa Biomedicals and used for cloning, were grown in Luria-Bertani (LB) or Terrific Broth medium with 50 ␮g/ml ampicillin when required. Native respiratory complex II was purified from the membrane fraction of S. tokodaii strain 7 The entire gene sequence of the S. tokodaii respiratory complex II operon sdhABCD has been determined [13] and deposited in the DNA Data Bank of Japan (DDBJ) (DDBJ accession number AB055061). The sequence determination was performed by the dideoxy chain termination method with an automatic DNA sequencer, ABI model 373A and 370A (Applied Biosystems Inc.). The PCR was carried out to amplify the sdhC gene coding for the archaeal SdhC subunit, using the S. tokodaii strain 7 genomic DNA and the following oligonucleotide primers (designed based on the nucleotide sequence AB055061 [13]): SdhC-N primer, 5Ј-GGG CCC GCT AGC ATG AGC TAT GCG TAT TAT-3Ј and SdhC-C primer, 5Ј-GGG CCC CTC

Novel Center C in Archaeal Respiratory Complex II

RESULTS AND DISCUSSION

TABLE I Curve fitting results for iron EXAFS