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Several intragenic mutations suppress theC. elegans isp-1(qm150)allele of the mitochondrial Rieske iron-sulfur protein (ISP), a catalytic subunit of Complex III of the respiratory chain. These mutations were located in a helical region of the “tether” span of ISP-1, distant from the primary mutation in the extrinsic head, and suppressed all pleiotropic phenotypes associated with theqm150allele. Analysis of these suppressors revealed control of electron transfer into Complex III through a “spring-loaded” mechanism involving a binding force for formation of enzyme-substrate complex, counter balanced by forces (a chemical “spring”) favoring helix formation in the tether. The primary P→S mutation results in inhibition of electron flow into the Q-cycle by decreasing the binding force, and the tether mutations relieve this inhibition by weakening the “spring.” In this commentary we discuss additional control features, and relate the primary inhibition to outcomes at the organismal level. In particular, the sensitivity to hyperoxia and the elevated reactive oxygen species (ROS) seen inisp-1(qm150), likely reflect over-reduction of the quinone pool, which is upstream of the inhibited site; at high O2, this would lead to increased ROS production through complex I. We speculate that alternative NADH:ubiquinone oxidoreductase activity inC. elegansfrom the worm apoptosis inducing factor (AIF) homolog (WAH-1) might also be involved, and that WAH-1 might have a “canary” function in detection of this adverse state (high O2/reduced pool), and a role in protection of the organism by transformation to AIF-like products, and apoptotic recycling of defective cells. [ABSTRACT FROM AUTHOR]

Abstract: We re-examine the pH dependence of partial processes of ubihydroquinone (QH2) turnover in Glu-295 mutants in Rhodobacter sphaeroides to clarify the mechanistic role. In more crippled mutants, the bell-shaped pH profile of wildtype was replaced by dependence on a single pK at ~8.5 favoring electron transfer. Loss of the pK at 6.5 reflects a change in the rate-limiting step from the first to the second electron transfer. Over the range of pH 6–8, no major pH dependence of formation of the initial reaction complex was seen, and the rates of bypass reactions were similar to the wildtype. Occupancy of the Qo-site by semiquinone (SQ) was similar in the wildtype and the Glu→Trp mutant. Since heme b L is initially oxidized in the latter, the bifurcated reaction can still occur, allowing estimation of an empirical rate constant <103 s−1 for reduction of heme b L by SQ from the domain distal from heme b L, a value 1000-fold smaller than that expected from distance. If the pK ~8.5 in mutant strains is due to deprotonation of the neutral semiquinone, with Q•− as electron donor to heme b L, then in wildtype this low value would preclude mechanisms for normal flux in which semiquinone is constrained to this domain. A kinetic model in which Glu-295 catalyzes H+ transfer from QH•, and delivery of the H+ to exit channel(s) by rotational displacement, and facilitates rapid electron transfer from SQ to heme b L by allowing Q•− to move closer to the heme, accounts well for the observations. [Copyright &y& Elsevier]

Abstract: The homodimeric bc 1 complexes are membrane proteins essential in respiration and photosynthesis. The ~11Å distance between the two b L-hemes of the dimer opens the possibility of electron transfer between them, but contradictory reports make such inter-monomer electron transfer controversial. We have constructed in Rhodobacter sphaeroides a heterodimeric expression system similar to those used before, in which the bc 1 complex can be mutated differentially in the two copies of cyt b to test for inter-monomer electron transfer, but found that genetic recombination by cross-over then occurs to produce wild-type homodimer. Selection pressure under photosynthetic growth always favored the homodimer over heterodimeric variants enforcing inter-monomer electron transfer, showing that the latter are not competitive. These results, together with kinetic analysis of myxothiazol titrations, demonstrate that inter-monomer electron transfer does not occur at rates competitive with monomeric turnover. We examine the results from other groups interpreted as demonstrating rapid inter-monomer electron transfer, conclude that similar mechanisms are likely to be in play, and suggest that such claims might need to be re-examined. [Copyright &y& Elsevier]

Mitochondria play an important role in numerous diseases as well as normative aging. Severe reduction in mitochondrial function contributes to childhood disorders such as Leigh Syndrome, whereas mild disruption can extend the lifespan of model organisms. The Caenorhabditis elegans isp-1 gene encodes the Rieske iron-sulfur protein subunit of cytochrome c oxidoreductase (complex III of the electron transport chain). The partial loss of function allele, isp-1(qm150), leads to several pleiotropic phenotypes. To better understand the molecular mechanisms of ISP-1 function, we sought to identify genetic suppressors of the delayed development of isp-1(qm150) animals. Here we report a series of intragenic suppressors, all located within a highly conserved six amino acid tether region of ISP-1. These intragenic mutations suppress all of the evaluated isp-1(qm150) phenotypes, including developmental rate, pharyngeal pumping rate, brood size, body movement, activation of the mitochondrial unfolded protein response reporter, CO2 production, mitochondrial oxidative phosphorylation, and lifespan extension. Furthermore, analogous mutations show a similar effect when engineered into the budding yeast Rieske iron-sulfur protein Rip1, revealing remarkable conservation of the structure-function relationship of these residues across highly divergent species. The focus on a single subunit as causal both in generation and in suppression of diverse pleiotropic phenotypes points to a common underlying molecular mechanism, for which we propose a "spring-loaded" model. These observations provide insights into how gating and control processes influence the function of ISP-1 in mediating pleiotropic phenotypes including developmental rate, movement, sensitivity to stress, and longevity. [ABSTRACT FROM AUTHOR]

The rate-determining step in the overall turnover of the bc1 complex is electron transfer from ubiquinol to the Rieske iron-sulfur protein (ISP) at the Qo-site. Structures of the ISP from Rhodobactersphaeroides show that serine 154 and tyrosine 156 form H-bonds to S-1 of the [2Fe-2S] cluster and to the sulfur atom of the cysteine liganding Fe-1 of the cluster, respectively. These are responsible in part for the high potential (Em,7 ∼300 mV) and low PKa (7.6) of the ISP, which determine the overall reaction rate of the bc1 complex. We have made site-directed mutations at these residues, measured thermodynamic properties using protein film voltammetry to evaluate the Em and PKa values of ISPs, explored the local proton environment through two-dimensional electron spin echo envelope modulation, and characterized function in strains S154T, S154C, S154A, Y156F, and Y156W. Alterations in reaction rate were investigated under conditions in which concentration of one substrate (ubiquinol or ISPox) was saturating and the other was varied, allowing calculation of kinetic terms and relative affinities. These studies confirm that H-bonds to the cluster or its ligands are important determinants of the electrochemical characteristics of the ISP, likely through electron affinity of the interacting atom and the geometry of the H-bonding neighborhood. The calculated parameters were used in a detailed Marcus-Bronsted analysis of the dependence of rate on driving force and pH. The proton-first-then-electron model proposed accounts naturally for the effects of mutation on the overall reaction. [ABSTRACT FROM AUTHOR]