Abstract
The mitochondrial bc(1) complex catalyzes the oxidation of ubiquinol and the reduction of cytochrome (cyt) c. The cyt b mutation A144F has been introduced in yeast by the biolistic method. This residue is located in the cyt b cd(1) amphipathic helix in the quinol-oxidizing (Q(O)) site. The resulting mutant was respiration-deficient and was affected in the quinol binding and electron transfer rates at the Q(O) site. An intragenic suppressor mutation was selected (A144F+F179L) that partially alleviated the defect of quinol oxidation of the original mutant A144F. The suppressor mutation F179L, located at less than 4 A from A144F, is likely to compensate directly the steric hindrance caused by phenylalanine at position 144. A second set of suppressor mutations was obtained, which also partially restored the quinol oxidation activity of the bc(1) complex. They were located about 20 A from A144F in the hinge region of the iron-sulfur protein (ISP) between residues 85 and 92. This flexible region is crucial for the movement of the ISP between cyt b and cyt c(1) during enzyme turnover. Our results suggested that the compensatory effect of the mutations in ISP was due to the repositioning of this subunit on cyt b during quinol oxidation. This genetic and biochemical study thus revealed the close interaction between the cyt b cd(1) helix in the quinol-oxidizing Q(O) site and the ISP via the flexible hinge region and that fine-tuning of the Q(O) site catalysis can be achieved by subtle changes in the linker domain of the ISP.
Highlights
The membrane-bound mitochondrial bc1 complex (b6f complex in chloroplasts and cyanobacteria) is a key component of the respiratory and photosynthetic electron transfer chains
Our results suggested that the compensatory effect of the mutations in ironsulfur protein (ISP) was due to the repositioning of this subunit on cyt b during quinol oxidation
From bacteria to higher eukaryotes, contain three subunits forming the catalytic core of the enzyme and carrying four redox centers as follows: the iron-sulfur protein (ISP)1 with a [2Fe-2S] cluster; the monohemic cyt c1; the dihemic mitochondrially encoded cytochrome b with a low potential bL heme (Em7 around Ϫ50 mV, where Em indicates equilibrium redox midpoint potential) located near the positive side of the membrane; and a high potential bH heme (Em7 around ϩ90 mV) located on the negative side of the membrane
Summary
The membrane-bound mitochondrial bc complex (b6f complex in chloroplasts and cyanobacteria) is a key component of the respiratory and photosynthetic electron transfer chains (see Refs. 1–5 for reviews). 19), and more recently, the yeast bc complex structure was obtained at 2.3 Å, including bound water molecules (20) These different structures as well as biochemical and spectroscopic data (21–29) suggest that the extrinsic carboxyl-terminal domain of the ISP carrying the [2Fe-2S] cluster moves between a position close to cyt b (proximal conformation or “b” state) and a position close to cyt c1 (distal conformation or “c1” state), solving the apparent incompatibility between distances and rate of electron transfer between the redox centers (16 –19). The abbreviations used are: ISP, iron-sulfur protein; RIP, nuclear gene coding for the iron-sulfur protein; cyt, cytochrome; bL, low potential cyt b heme; bH, high potential cyt b heme; DB, 2.3-dimethoxy-5methyl-6-decyl-1.4-benzoquinone; DBH2, reduced form of DB; QO, ubiquinol oxidation site on the positive side of the inner mitochondrial membrane; QI, ubiquinone reduction site on the negative side of the inner mitochondrial membrane; WT, parental strain; MOPS, 4-morpholinepropanesulfonic acid
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