The road to the crystal structure of the cytochrome bc1 complex from the anoxigenic, photosynthetic bacterium Rhodobacter sphaeroides.

The advantages of using bacterial systems to study the mechanism and function of cytochrome bc (1) complexes do not extend readily to their structural investigations. High quality crystals of bacterial complexes have been difficult to obtain despite the enzymes' smaller sizes and simpler subunit compositions compared to their mitochondrial counterparts. In the course of the structure determination of the bc (1) complex from R. sphaeroides, we observed that the growth of only low quality crystals correlated with low activity and stability of the purified complex, which was mitigated in part by introducing a double mutations to the enzyme. The S287R(cyt b)/V135S(ISP) mutant shows 40% increase in electron transfer activity and displays a 4.3 degrees C increase in thermal stability over wild-type enzyme. The amino acid histidine was found important in maintaining structural integrity of the bacterial complex, while the respiratory inhibitors such as stigmatellin are required for immobilization of the iron-sulfur protein extrinsic domain. Crystal quality of the R. sphaeroides bc (1) complex can be improved further by the presence of strontium ions yielding crystals that diffracted X-rays to better than 2.3 A resolution. The improved crystal quality can be understood in terms of participation of strontium ions in molecular packing arrangement in crystal.

Inhibitor-complexed structures of the cytochrome bc1 from the photosynthetic bacterium Rhodobacter sphaeroides.

The cytochrome bc(1) complex (bc(1)) is a major contributor to the proton motive force across the membrane by coupling electron transfer to proton translocation. The crystal structures of wild type and mutant bc(1) complexes from the photosynthetic purple bacterium Rhodobacter sphaeroides (Rsbc(1)), stabilized with the quinol oxidation (Q(P)) site inhibitor stigmatellin alone or in combination with the quinone reduction (Q(N)) site inhibitor antimycin, were determined. The high quality electron density permitted assignments of a new metal-binding site to the cytochrome c(1) subunit and a number of lipid and detergent molecules. Structural differences between Rsbc(1) and its mitochondrial counterparts are mostly extra membranous and provide a basis for understanding the function of the predominantly longer sequences in the bacterial subunits. Functional implications for the bc(1) complex are derived from analyses of 10 independent molecules in various crystal forms and from comparisons with mitochondrial complexes.

Generation, characterization and crystallization of a highly active and stable cytochrome bc1 complex mutant from Rhodobacter sphaeroides.

The availability of the three dimensional structure of mitochondrial enzyme, obtained by X-ray crystallography, allowed a significant progress in the understanding of the structure-function relation of the cytochrome bc(1) complex. Most of the structural information obtained has been confirmed by molecular genetic studies of the bacterial complex. Despite its small size and simple subunit composition, high quality crystals of the bacterial complex have been difficult to obtain and so far, only low resolution structural data has been reported. The low quality crystal observed is likely associated in part with the low activity and stability of the purified complex. To mitigate this problem, we recently engineered a mutant [S287R(cytb)/V135S(ISP)] from Rhodobacter sphaeroides to produce a highly active and more stable cytochrome bc(1) complex. The purified mutant complex shows a 40% increase in electron transfer activity as compared to that of the wild type enzyme. Differential scanning calorimetric study shows that the mutant is more stable than the wild type complex as indicated by a 4.3 degrees C increase in the thermo-denaturation temperature. Crystals formed from this mutant complex, in the presence of stigmatellin, diffract X-rays up to 2.9 Angstroms resolution.

The disulfide bridge in the head domain of rhodobacter sphaeroides cytochrome c1 is needed to maintain its structural integrity.

Cytochrome c(1) of Rhodobacter sphaeroides ubiquinol-cytochrome c oxidoreductase contains several insertions and deletions that distinguish it from the complex of other higher organisms. Additionally, this bacterial cytochrome c(1) contains two nonconserved cysteines, C145 and C169, with the latter included in the second long insertion located upstream of the sixth heme ligand, M185. The orientation of the insertions and the state of these non-heme binding cysteines remain unknown. Mutating one or both cysteines is found to have comparable effects on the functionality of the cytochrome bc(1) complex. Mutants show an electron transfer activity decreased to a rate that is still high enough to support delayed photosynthetic growth. The mutated cytochrome c(1) has a decreased E(m) without any alteration in the heme ligation environment since none of the mutants binds carbon monoxide. The low E(m) is believed to be caused by a structural modification in the head domain of cytochrome c(1). Analysis of the mutants reveals that the two cysteines form a disulfide bridge. Cleavage of cytochrome c(1) between the two cysteines followed by gel electrophoresis shows two fragments only under reducing conditions, confirming the existence of a disulfide bridge. The disulfide bridge is essential in maintaining the structural integrity of cytochrome c(1) and thus the functionality of the cytochrome bc(1) complex.

Crystallographic studies of quinol oxidation site inhibitors: a modified classification of inhibitors for the cytochrome bc(1) complex.

Cytochrome bc(1) is an integral membrane protein complex essential for cellular respiration and photosynthesis; it couples electron transfer from quinol to cytochrome c to proton translocation across the membrane. Specific bc(1) inhibitors have not only played crucial roles in elucidating the mechanism of bc(1) function but have also provided leads for the development of novel antibiotics. Crystal structures of bovine bc(1) in complex with the specific Q(o) site inhibitors azoxystrobin, MOAS, myxothiazol, stigmatellin and 5-undecyl-6-hydroxy-4,7-dioxobenzothiazole were determined. Interactions, conformational changes and possible mechanisms of resistance, specific to each inhibitor, were defined. Residues and secondary structure elements that are capable of discriminating different classes of Q(o) site inhibitors were identified for the cytochrome b subunit. Directions in the displacement of the cd1 helix of cytochrome b subunit in response to various Q(o) site inhibitors were correlated to the binary conformational switch of the extrinsic domain of the iron-sulfur protein subunit. The new structural information, together with structures previously determined, provide a basis that, combined with biophysical and mutational data, suggest a modification to the existing classification of bc(1) inhibitors. bc(1) inhibitors are grouped into three classes: class P inhibitors bind to the Q(o) site, class N inhibitors bind to the Q(i) site and the class PN inhibitors target both sites. Class P contains two subgroups, Pm and Pf, that are distinct by their ability to induce mobile or fixed conformation of iron-sulfur protein.