Jonathan L. Cape

A semiquinone intermediate generated at the Qo site of the cytochrome bc1 complex: Importance for the Q-cycle and superoxide production

The cytochrome bc1 and related complexes are essential energy-conserving components of mitochondrial and bacterial electron transport chains. They orchestrate a complex sequence of electron and proton transfer reactions resulting in the oxidation of quinol, the reduction of a mobile electron carrier, and the translocation of protons across the membrane to store energy in an electrochemical proton gradient. The enzyme can also catalyze substantial rates of superoxide production, with deleterious physiological consequences. Progress on understanding these processes has been hindered by the lack of observable enzymatic intermediates. We report the first direct detection of a semiquinone radical generated by the Qo site using continuous wave and pulsed EPR spectroscopy. The radical is a ubisemiquinone anion and is sensitive to both specific inhibitors and mutations within the Qo site as well as O2, suggesting that it is the elusive intermediate responsible for superoxide production. Paramagnetic interactions show that the new semiquinone species is buried in the protein, probably in or near the Qo site but not strongly interacting with the 2Fe2S cluster. The semiquinone is substoichiometric, even with conditions optimized for its accumulation, consistent with recently proposed models where the semiquinone is destabilized to limit superoxide production. The discovery of this intermediate provides a critical tool to directly probe the elusive chemistry that takes place within the Qo site.

A Porous Metal−Organic Replica of α-PbO2 for Capture of Nerve Agent Surrogate

A novel metal-organic replica of α-PbO(2) exhibits high capacity for capture of nerve agent surrogate.

Q-Cycle Bypass Reactions at the Qo Site of the Cytochrome bc1 (and Related) Complexes

Publisher Summary This chapter presents an overview of Q-cycle bypass reactions at the Q 0 site of the cytochrome bc 1 and related complexes. The availability of high-resolution crystal structures, a large array of mutant strains in a number of species, and new spectroscopic approaches should greatly facilitate elucidation of the mechanisms by which the various bypass reactions are prevented. To aid this approach, this chapter describes the application and limitations of assays for detection of the various bypass reactions. A method is described for probing conformational changes in the ISP head domain, which are thought to be critical for preventing some of the bypass reactions. The chapter describes the Q-cycle and its bypass reactions. The chapter also elaborates about estimating the concentrations of cyt bc 1 and b6f complexes. The chapter presents the estimates of cyt bc 1 and b6f concentrations and measurements of the bifurcated oxidation of QH2 and probes the involvement of ISP domain movements in restricting bypass reactions. A simple method for estimating changes in the orientation and ordering of membrane-bound anisotropic EPR signals is also presented in the chapter.

Substrate Redox Potential Controls Superoxide Production Kinetics in the Cytochrome bc Complex

The Q-cycle mechanism of the cytochrome bc(1) complex maximizes energy conversion during the transport of electrons from ubiquinol to cytochrome c (or alternate physiological acceptors), yet important steps in the Q-cycle are still hotly debated, including bifurcated electron transport, the high yield and specificity of the Q-cycle despite possible short-circuits and bypass reactions, and the rarity of observable intermediates in the oxidation of quinol. Mounting evidence shows that some bypass reactions producing superoxide during oxidation of quinol at the Q(o) site diverge from the Q-cycle rather late in the bifurcated reaction and provide an additional means of studying initial reactions of the Q-cycle. Bypass reactions offer more scope for controlling and manipulating reaction conditions, e.g., redox potential, because they effectively isolate or decouple the Q-cycle initial reactions from later steps, preventing many complications and interactions. We examine the dependence of oxidation rate on substrate redox potential in the yeast cytochrome bc(1) complex and find that the rate limitation occurs at the level of direct one-electron oxidation of quinol to semiquinone by the Rieske protein. Oxidation of semiquinone and reduction of cyt b or O(2) are subsequent, distinct steps. These experimental results are incompatible with models in which the transfer of electrons to the Rieske protein is not a distinct step preceding transfer of electrons to cytochrome b, and with conformational gating models that produce superoxide by different rate-limiting reactions from the normal Q-cycle.