Direct Demonstration of Half-of-the-sites Reactivity in the Dimeric Cytochrome bc1 Complex

Abstract

We previously proposed that the dimeric cytochrome bc 1 complex exhibits half-of-the-sites reactivity for ubiquinol oxidation and rapid electron transfer between bc 1 monomers (Covian, R., Kleinschroth, T., Ludwig, B., and Trumpower, B. L. (2007) J. Biol. Chem. 282, 22289 -22297). Here, we demonstrate the previously proposed half-of-the-sites reactivity and intermonomeric electron transfer by characterizing the kinetics of ubiquinol oxidation in the dimeric bc 1 complex from Paracoccus denitrificans that contains an inactivating Y147S mutation in one or both cytochrome b subunits. The enzyme with a Y147S mutation in one cytochrome b subunit was catalytically fully active, whereas the activity of the enzyme with a Y147S mutation in both cytochrome b subunits was only 10 -16% of that of the enzyme with fully wild-type or heterodimeric cytochrome b subunits. Enzyme with one inactive cytochrome b subunit was also indistinguishable from the dimer with two wild-type cytochrome b subunits in rate and extent of reduction of cytochromes b and c 1 by ubiquinol under pre-steady-state conditions in the presence of antimycin. However, the enzyme with only one mutated cytochrome b subunit did not show the stimulation in the steady-state rate that was observed in the wildtype dimeric enzyme at low concentrations of antimycin, confirming that the half-of-the-sites reactivity for ubiquinol oxidation can be regulated in the wild-type dimer by binding of inhibitor to one ubiquinone reduction site.The cytochrome bc 1 complex is a multisubunit enzyme that generates a transmembrane protonmotive force by transferring electrons from ubiquinol to cytochrome c. Energy conservation in this enzyme complex is ensured by the oxidation of ubiquinol and the reduction of ubiquinone at active sites located on opposite sides of the membrane, as described by the protonmotive Q-cycle (1). High resolution structures have shown that the bc 1 complex is a dimer composed of two copies of cytochrome b, the Rieske iron-sulfur protein, and cytochrome c 1 , which are the only polypeptides present in some bacterial enzymes (2), in addition to six to eight additional subunits present exclusively in each monomer of the eukaryotic complex (3-5).The functional relevance of this dimeric arrangement has been supported by an extensive body of kinetic evidence (for review, see Ref. 6), which includes the key observations that only one ubiquinol oxidation site, or center P, is active when the two ubiquinone reduction, or center N, sites are occupied by inhibitors (7), that electrons rapidly equilibrate between the cytochrome b subunits (8, 9), and that there is conformational communication between center P and center N sites (10). We have proposed that this half-of-the sites activity at center P also exists under normal steady-state conditions in the absence of inhibitors and that this mechanism minimizes the leakage of electrons to oxygen under conditions that would favor the accumulation of electrons in the cytochrome b hemes (8, 11). Evidence consistent with this...

Role of the -PEWY-glutamate in catalysis at the Qo-site of the Cyt bc1 complex

“…A kinetic pattern similar to that on the first flash, - full reduction of heme b H , and partial reduction (~50%, depending on pH) of heme c 1 , - is seen on reduction of isolated oxidized complex by decyl-UQ in the presence of antimycin [31, 53, 86–88], and is well accounted for by the modified Q-cycle through consideration of the constraints discussed above. However, in experiments with the mitochondrial complex, the behavior has recently been interpreted differently.…”

“…However, in experiments with the mitochondrial complex, the behavior has recently been interpreted differently. An ingenious mechanism proposed by Trumpower and colleagues [31, 53, 86–88] borrows the bifurcated reaction from the Q-cycle, but introduces control by an unspecified mechanism that operates within the dimer to limit Q o -site activity to only one of the two monomers, giving a half-of-sites functionality. To explain the poise of reactants discussed above, it is then necessary to postulate that the active Q o -site can access its own high potential chain, but both low potential chains through inter-monomer electron transfer at the level of heme b L .…”

“…Although several publications have raised objections to specific features of the mechanism proposed [31, 38, 53–56], some support for the proton exit scenario was provided by studies of the role of E295 in Zn-binding and inhibition [52]. In this paper, we have mutated the - PEWY-glutamate to D, G, Q, W, K, and L, and explored the effects on the bifurcated reaction and bypass processes, and their pH dependence compared to wildtype.…”

Electronic Connection Between the Quinone and CytochromecRedox Pools and Its Role in Regulation of Mitochondrial Electron Transport and Redox Signaling

“…For cytochrome bc 1 , this concerns cytochrome b subunit which is coded by a gene located in mtDNA. Given that this gene does not differentiate between monomers and that mtDNA is present in several copies in mitochondria, the presence of mutations in just a part of the copies of the gene is likely to result in expression of both symmetrically and asymmetrically mutated dimers of cytochrome bc 1 (FIGURE 9) as observed in bacterial two-plasmid model systems (45,141,151). Thus it can be anticipated that the amount of the asymmetrically damaged complexes increases with age.…”

Distinguishing Protonation States of Histidine Ligands to the Oxidized Rieske Iron–Sulfur Cluster through 15N Vibrational Frequency Shifts

“…[8] The bc1 complex is ac omponent of the electron-transport chain and functions by transporting electrons through the oxidation of ubiquinol to ubiquinone, while simultaneously translocating protons across the cellular membrane. [9][10][11] The chloroplast homologue of cytochromebc1 is cytochrome b6f, which mediates electron transfer between PhotosystemsI and II. [12][13][14] In both complexes, as eries of oxidations and reductions create an electrochemical gradient, which is later used to facilitate the production of ATP by the enzyme ATPs ynthase.…”