Mutations in cytochrome b that affect kinetics of the electron transfer reactions at center N in the yeast cytochrome bc1 complex

AbstractWe have examined the pre-steady-state kinetics and thermodynamic properties of the b hemes in variants of the yeast cytochrome bc1 complex that have mutations in the quinone reductase site (center N). Trp-30 is a highly conserved residue, forming a hydrogen bond with the propionate on the high potential b heme (bH heme). The substitution by a cysteine (W30C) lowers the redox potential of the heme and an apparent consequence is a lower rate of electron transfer between quinol and heme at center N. Leu-198 is also in close proximity to the bH heme and a L198F mutation alters the spectral properties of the heme but has only minor effects on its redox properties or the electron transfer kinetics at center N. Substitution of Met-221 by glutamine or glutamate results in the loss of a hydrophobic interaction that stabilizes the quinone ligands. Ser-20 and Gln-22 form a hydrogen-bonding network that includes His-202, one of the carbonyl groups of the ubiquinone ring, and an active-site water. A S20T mutation has long-range structural effects on center P and thermodynamic effects on both b hemes. The other mutations (M221E, M221Q, Q22E and Q22T) do not affect the ubiquinol oxidation kinetics at center P, but do modify the electron transfer reactions at center N to various extents. The pre-steady reduction kinetics suggest that these mutations alter the binding of quinone ligands at center N, possibly by widening the binding pocket and thus increasing the distance between the substrate and the bH heme. These results show that one can distinguish between the contribution of structural and thermodynamic factors to center N function

Disulfide Bond Formation Involves a Quinhydrone-Type Charge–Transfer Complex

The chemistry of disulfide exchange in biological systems is well studied. However, the detailed mechanism of how oxidizing equivalents are derived to form disulfide bonds in proteins is not clear. In prokaryotic organisms, it is known that DsbB delivers oxidizing equivalents through DsbA to secreted proteins. DsbB becomes reoxidized by reducing quinones that are part of the membrane-bound electron-transfer chains. It is this quinone reductase activity that links disulfide bond formation to the electron transport system. We show here that purified DsbB contains the spectral signal of a quinhydrone, a charge-transfer complex consisting of a hydroquinone and a quinone in a stacked configuration. We conclude that disulfide bond formation involves a stacked hydroquinone-benzoquinone pair that can be trapped on DsbB as a quinhydrone charge-transfer complex. Quinhydrones are known to be redox-active and are commonly used as redox standards, but, to our knowledge, have never before been directly observed in biological systems. We also show kinetically that this quinhydrone-type charge-transfer complex undergoes redox reactions consistent with its being an intermediate in the reaction mechanism of DsbB. We propose a simple model for the action of DsbB where a quinhydrone-like complex plays a crucial role as a reaction intermediate

Exchangeability of Qsr1p, a large ribosomal subunit protein required for subunit joining, suggests a novel translational regulatory mechanism

AbstractQsr1p is a 60S ribosomal subunit protein that is necessary for joining of large and small ribosomal subunits and is also one of the last proteins assembled onto the 60S ribosomal subunit in the cytoplasm. The finding that Qsr1p is identical to L7, a protein previously shown to cycle on and off large ribosomal subunits in the cytoplasm, suggests that the addition of Qsr1p onto the 60S ribosomal subunit could be utilized as a translational regulatory mechanism by limiting the supply of functional 60S subunits

The cleaved presequence is not required for import of subunit 6 of the cytochrome bc 1 complex into yeast mitochondria or assembly into the complex*

Abstract Subunit 6 of the yeast cytochrome bc 1 complex contains a 25 amino acid presequence that is not present in the mature form of the protein in the bc 1 complex. The presequence of subunit 6 is atypical of presequences responsible for targeting proteins to mitochondria. Whereas mitochondrial targeting sequences rarely contain acidic residues and typically contain basic residues that can potentially form an amphiphilic structure, the presequence of subunit 6 contains only one basic amino acid and is enriched in acidic amino acids. If the 25 amino acid presequence is deleted, subunit 6 is imported into mitochondria and assembled into the cytochrome bc 1 complex and the activity of the bc 1 complex is identical to that from a wild-type yeast strain. However, if the C-terminal 45 amino acids are truncated from the protein, subunit 6 is not present in the mitochondria and the activity of the bc 1 complex is diminished by half, identical to that of the bc 1 complex from a yeast strain in which the QCR6 gene is deleted. These results indicate that the presequence of subunit 6 is not required for targeting to mitochondria or assembly of the subunit into the bc 1 complex and that information necessary for targeting and import into mitochondria may be present in the C-terminus of the protein. z 1999 Federation of European Biochemical Societies

Parameters determining the relative efficacy of hydroxy-naphthoquinone inhibitors of the cytochrome bc1 complex

Hydroxy-naphthoquinones are competitive inhibitors of the cytochrome bc1 complex that bind to the ubiquinol oxidation site between cytochrome b and the iron-sulfur protein and presumably mimic a transition state in the ubiquinol oxidation reaction catalyzed by the enzyme. The parameters that affect efficacy of binding of these inhibitors to the bc1 complex are not well understood. Atovaquone®, a hydroxy-naphthoquinone, has been used therapeutically to treat Pneumocystis carinii and Plasmodium infections. As the pathogens have developed resistance to this drug, it is important to understand the molecular basis of the drug resistance and to develop new drugs that can circumvent the drug resistance. We previously developed the yeast and bovine bc1 complexes as surrogates to model the interaction of atovaquone with the bc1 complexes of the target pathogens and human host

Cytochrome b Mutations That Modify the Ubiquinol-binding Pocket of the Cytochrome bc 1 Complex and Confer Anti-malarial Drug Resistance in Saccharomyces cerevisiae

Atovaquone is a new anti-malarial agent that specifically targets the cytochrome bc1 complex and inhibits parasite respiration. A growing number of failures of this drug in the treatment of malaria have been genetically linked to point mutations in the mitochondrial cytochrome b gene. To better understand the molecular basis of atovaquone resistance in malaria, we introduced five of these mutations, including the most prevalent variant found in Plasmodium falciparum (Y268S), into the cytochrome b gene of the budding yeast Saccharomyces cerevisiae and thus obtained cytochrome bc1 complexes resistant to inhibition by atovaquone. By modeling the variations in cytochrome b structure and atovaquone binding with the mutated bc1 complexes, we obtained the first quantitative explanation for the molecular basis of atovaquone resistance in malaria parasites

Molecular Basis for Atovaquone Binding to the Cytochrome bc 1 Complex

Atovaquone is a substituted 2-hydroxynaphthoquinone that is used therapeutically to treat Plasmodium falciparum malaria, Pneumocystis carinii pneumonia, and Toxoplasma gondii toxoplasmosis. It is thought to act on these organisms by inhibiting the cytochrome bc1 complex. We have examined the interaction of atovaquone with the bc1 complex isolated from Saccharomyces cerevisiae, a surrogate, nonpathogenic fungus. Atovaquone inhibits the bc1 complex competitively with apparent Ki = 9 nm, raises the midpoint potential of the Rieske iron-sulfur protein from 285 to 385 mV, and shifts the g values in the EPR spectrum of the Rieske center. These results indicate that atovaquone binds to the ubiquinol oxidation pocket of the bc1 complex, where it interacts with the Rieske iron-sulfur protein. A computed energy-minimized structure for atovaquone liganded to the yeast bc1 complex suggests that a phenylalanine at position 275 of cytochrome b in the bovine bc1 complex, as opposed to leucine at the equivalent position in the yeast enzyme, is responsible for the decreased sensitivity of the bovine bc1 complex (Ki = 80 nm) to atovaquone. When a L275F mutation was introduced into the yeast cytochrome b, the sensitivity of the yeast enzyme to atovaquone decreased (Ki = 100 nm) with no loss in activity, confirming that the L275F exchange contributes to the differential sensitivity of these two species to atovaquone. These results provide the first molecular description of how atovaquone binds to the bc1 complex and explain the differential inhibition of the fungal versus mammalian enzymes

A concerted, alternating sites mechanism of ubiquinol oxidation by the dimeric cytochrome bc1 complex, Biochim. Biophys. Acta 1555

Abstract A refinement of the protonmotive Q cycle mechanism is proposed in which oxidation of ubiquinol is a concerted reaction and occurs by an alternating, half-of-the-sites mechanism. A concerted mechanism of ubiquinol oxidation is inferred from the finding that there is reciprocal control between the high potential and low potential redox components involved in ubiquinol oxidation. The potential of the Rieske ironsulfur protein controls the rate of reduction of the b cytochromes, and the potential of the b cytochromes controls the rate of reduction of the Rieske protein and cytochrome c 1 . A concerted mechanism of ubiquinol oxidation reconciles the findings that the ubiquinol -cytochrome c reductase kinetics of the bc 1 complex include both a pH dependence and a dependence on Rieske iron -sulfur protein midpoint potential. An alternating, half-of-the-sites mechanism for ubiquinol oxidation is inferred from the finding that some inhibitory analogs of ubiquinol that block ubiquinol oxidation by binding to the ubiquinol oxidation site in the bc 1 complex inhibit the yeast enzyme with a stoichiometry of 0.5 per bc 1 complex. One molecule of inhibitor is sufficient to fully inhibit the dimeric enzyme, and the binding is anti-cooperative, in that a second molecule of inhibitor binds with much lower affinity to a dimer in which an inhibitor molecule is already bound. An alternating, half-of-the-sites mechanism implies that, at least under some conditions, only half of the sites in the dimeric enzyme are reactive at any one time. This provides a raison d'être for the dimeric structure of the enzyme, in that bc 1 activity may be regulated and capable of switching between a half-of-the-sites active and a fully active enzyme.