The structure-function interface in the cytochrome bc1 complex family

Mitochondrial bc1 complexes and chloroplast b6f complexes belong to a common family of quinol oxidizing enzymes. We have made a systematic survey of the redox subunits of the bc-type quinol oxidases for all known bacterial sequences and have identified six new representatives of the enzyme. Sequences of the subunits containing the conserved ligands indicating either an iron-sulfur protein (ISP), two b-hemes (cyt b) subunit, or a c-heme (cyt c) were used to study the phylogeny of each of these polypeptides. Cyt b subunit of the bc1 complex from Rhodobacter sphaeroides was modified to introduce two distinctive features of b 6f complexes. The stability of the semiquinone formed at the quinone-reduction site was decreased when cyt b was split into two polypeptides thus mimicking the organization of cyt b6 and subunit IV in the b6 f-complexes. Addition of an extra threonine residue between the histidine ligands of the two b-hemes in helix D resulted in modification of the spectrum and redox potential of the bL-heme. Mutations of Tyr-156, located close to the iron-sulfur cluster in the ISP to Leu, Phe or Trp led to alteration of the midpoint potential of the ISP. Slowing in the rate of quinol oxidation correlated with a modified redox potential suggesting that the first electron transfer from quinol might contribute to the activation barrier. The Trp mutation also introduced a modification in the pK value of the ISP which was reflected in the pH dependence of the rate of quinol oxidation. This strongly supports a model in which the dissociated form of the oxidized ISP is required for formation of the reaction complex. The fbcB and fbcC genes of the fbc-operon, encoding cyt b and c 1 of the bc1-complex were extended with a segment to encode a poly-histidine tag and this was introduced into Rb. sphaeroides. The properties of the transformants were essentially the same as those from wild-type strains. bc1-complex can be easily purified from these strains using a one step affinity column purification. We have applied the method of site-directed labeling to the bc1-complex of Rb. sphaeroides to study the conformational changes of the ISP. Using nitroxide spin labels or fluorophores, attached to genetically engineered cysteine residues we have determined biochemical conditions that may control such conformational changes under steady state conditions.