Identification of the heme axial ligands in the cytochrome b562 of the Saccharomyces cerevisiae succinate dehydrogenase.

Paul F. Yau,Bernard D. Lemire,Kayode S. Oyedotun

doi:10.1074/jbc.m311877200

Paul F. Yau, Bernard D. Lemire + Show 1 more

Open Access

https://doi.org/10.1074/jbc.m311877200

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Abstract

Succinate dehydrogenase (SDH) plays a key role in energy generation by coupling the oxidation of succinate to the reduction of ubiquinone in the mitochondrial electron transport chain. The Saccharomyces cerevisiae SDH is composed of a catalytic dimer of the Sdh1p and Sdh2p subunits containing flavin adenine dinucleotide (FAD) and iron-sulfur clusters and a heme b-containing membrane-anchoring domain comprised of the Sdh3p and Sdh4p subunits. We systematically mutated all the histidine and cysteine residues in Sdh3p and Sdh4p to identify the residues involved in axial heme ligation. The mutants were characterized for growth on a non-fermentable carbon source, for enzyme assembly, for succinate-dependent quinone reduction, for heme b content, and for heme spectral properties. Mutation of Sdh3p His-46 or His-113 leads to a marked reduction in the catalytic efficiency of the enzyme for quinone reduction, suggesting that these residues form part of a quinone-binding site. We identified Sdh3p His-106 and Sdh4p Cys-78 as the most probable axial ligands for cytochrome b(562). Replacement of His-106 or Cys-78 with an alanine residue leads to a marked reduction in cytochrome b(562) content and to altered heme spectral characteristics that are consistent with a direct perturbation of heme b environment. This is the first identification of a cysteine residue serving as an axial ligand for heme b in the SDH family of enzymes. Loss of cytochrome b(562) has no effect on enzyme assembly and quinone reduction; the role of the heme in enzyme structure and function is discussed.

Highlights

Energy generation in most eukaryotes depends on the activity of the mitochondrial respiratory chain (MRC).1 The MRC is composed of four multisubunit enzymes, complexes I-IV, that transfer electrons from NADH and succinate to molecular oxygen and capture the energy of substrate oxidation in the form of proton gradient, which is used for ATP synthesis by complex V
The Saccharomyces cerevisiae Succinate dehydrogenase (SDH) is composed of a catalytic dimer of the Sdh1p and Sdh2p subunits containing flavin adenine dinucleotide (FAD) and iron-sulfur clusters and a heme b-containing membrane-anchoring domain comprised of the Sdh3p and Sdh4p subunits
Choice of Amino Acid Residues Targeted for Mutagenesis— The crystal structures of the E. coli SDH [16] and the W. succinogenes fumarate reductases (FRDs) [18] have shown that the heme axial ligands are histidine residues

Summary

Amino acid

His-99 a Letters in bold indicate the mutated nucleotides. Mutant residue Alanine Alanine Aspartic acid Alanine Alanine Alanine Alanine Alanine Histidine Alanine Alanine axial ligand because a more bulky histidine could not be accommodated without distorting the protein backbone conformation [44]. We employ site-directed mutagenesis and spectroscopic and catalytic analysis to investigate these predictions. Our results strongly suggest that the yeast SDH differs from other family members by using a cysteine residue as an axial heme ligand

EXPERIMENTAL PROCEDURES

RESULTS

Turnover numberc

DISCUSSION

NADH oxidasee

Membrane sample

We have presented data that strongly support the roles of the