Nuclearly inherited diuron-resistant mutations conferring a deficiency in the NADH--or succinate--ubiquinone oxidoreductase activity in Saccharomyces cerevisiae.

In Saccharomyces cerevisiae, diuron, antimycin and myxothiazol block the respiratory pathway at the bc1 complex level. Nuclearly inherited mutations located at the DIU3 and DIU4 loci confer in vitro resistance to diuron and cross-resistance to antimycin and myxothiazol at the NADH oxidase level. The mutant strains do not exhibit diuron resistance at the quinol-cytochrome-c oxidoreductase level. Thus, the apparent resistance does not seem to be the result of a modification of the inhibitory sites. Instead, the quinone reduction rate was found to be altered in the mutant. The diu3 mutations lead to a deficiency of the NADH--ubiquinone oxidoreductase activity, and the diu4 mutations to a deficiency of the succinate--ubiquinone oxidoreductase activity. On the basis of the model of Kröger and Klingenberg, a decrease of quinone reduction could explain the resistance to the bc1 complex inhibitors. Thus, the apparent resistance to the bc1 complex inhibitors was found to be due to a modification of the electron transfer kinetics

First Evidence for Existence of an Uphill Electron Transfer through the bc(1) and NADH-Q Oxidoreductase Complexes of the Acidophilic Obligate Chemolithotrophic Ferrous Ion-Oxidizing Bacterium Thiobacillus ferrooxidans

The energy-dependent electron transfer pathway involved in the reduction of pyridine nucleotides which is required for CO(2) fixation to occur in the acidophilic chemolithotrophic organism Thiobacillus ferrooxidans was investigated using ferrocytochrome c as the electron donor. The experimental results show that this uphill pathway involves a bc(1) and an NADH-Q oxidoreductase complex functioning in reverse, using an electrochemical proton gradient generated by ATP hydrolysis. Based on these results, a model is presented to explain the balance of the reducing equivalent from ferrocytochrome c between the exergonic and endergonic electron transfer pathways

Apparent redundancy of electron transfer pathways via bc1 complexes and terminal oxidases in the extremophilic chemolithoautotrophic Acidithiobacillus ferrooxidans

AbstractAcidithiobacillus ferrooxidans is an acidophilic chemolithoautotrophic bacterium that can grow in the presence of either the weak reductant Fe2+, or reducing sulfur compounds that provide more energy for growth than Fe2+. We have previously shown that the uphill electron transfer pathway between Fe2+ and NAD+ involved a bc1 complex that functions only in the reverse direction [J. Bacteriol. 182, (2000) 3602]. In the present work, we demonstrate both the existence of a bc1 complex functioning in the forward direction, expressed when the cells are grown on sulfur, and the presence of two terminal oxidases, a bd and a ba3 type oxidase expressed more in sulfur than in iron-grown cells, besides the cytochrome aa3 that was found to be expressed only in iron-grown cells. Sulfur-grown cells exhibit a branching point for electron flow at the level of the quinol pool leading on the one hand to a bd type oxidase, and on the other hand to a bc1→ba3 pathway. We have also demonstrated the presence in the genome of transcriptionally active genes potentially encoding the subunits of a bo3 type oxidase. A scheme for the electron transfer chains has been established that shows the existence of multiple respiratory routes to a single electron acceptor O2. Possible reasons for these apparently redundant pathways are discussed

Surface-modulated motion switch: Capture and release of iron–sulfur protein in the cytochrome bc(1) complex

In the cytochrome bc(1) complex, the swivel motion of the iron–sulfur protein (ISP) between two redox sites constitutes a key component of the mechanism that achieves the separation of the two electrons in a substrate molecule at the quinol oxidation (Q(o)) site. The question remaining is how the motion of ISP is controlled so that only one electron enters the thermodynamically favorable chain via ISP. An analysis of eight structures of mitochondrial bc(1) with bound Q(o) site inhibitors revealed that the presence of inhibitors causes a bidirectional repositioning of the cd1 helix in the cytochrome b subunit. As the cd1 helix forms a major part of the ISP binding crater, any positional shift of this helix modulates the ability of cytochrome b to bind ISP. The analysis also suggests a mechanism for reversal of the ISP fixation when the shape complementarity is significantly reduced after a positional reorientation of the reaction product quinone. The importance of shape complementarity in this mechanism was confirmed by functional studies of bc(1) mutants and by a structure determination of the bacterial form of bc(1). A mechanism for the high fidelity of the bifurcated electron transfer is proposed

A mitochondrial cytochrome b mutation causing severe respiratory chain enzyme deficiency in humans and yeast.

Contains fulltext : 48890.pdf (publisher's version ) (Closed access)Whereas the majority of disease-related mitochondrial DNA mutations exhibit significant biochemical and clinical heterogeneity, mutations within the mitochondrially encoded human cytochrome b gene (MTCYB) are almost exclusively associated with isolated complex III deficiency in muscle and a clinical presentation involving exercise intolerance. Recent studies have shown that a small number of MTCYB mutations are associated with a combined enzyme complex defect involving both complexes I and III, on account of the fact that an absence of assembled complex III results in a dramatic loss of complex I, confirming a structural dependence between these two complexes. We present the biochemical and molecular genetic studies of a patient with both muscle and brain involvement and a severe reduction in the activities of both complexes I and III in skeletal muscle due to a novel mutation in the MTCYB gene that predicts the substitution (Arg318Pro) of a highly conserved amino acid. Consistent with the dramatic biochemical defect, Western blotting and BN-PAGE experiments demonstrated loss of assembled complex I and III subunits. Biochemical studies of the equivalent amino-acid substitution (Lys319Pro) in the yeast enzyme showed a loss of enzyme activity and decrease in the steady-state level of bc1 complex in the mutant confirming pathogenicity