Enzymes of the bc 1 complex family are central components of all the main energy transduction systems of the biosphere, and catalyze essentially the same reaction, the transfer of reducing equivalents from a quinol (ubihydroquinone, QH 2 , 1 in the classical bc 1 complexes) in the lipid phase to a higher potential acceptor protein in the aqueous phase. This electron transfer is coupled to transfer of 1 H ϩ /e Ϫ across the membrane. It is generally agreed that the complex functions through a Q-cycle mechanism (1-7). The coupling to proton transfer depends on a bifurcated reaction at the Q o -site of the complex, in which the two electrons from QH 2 are passed to two different chains. The initial acceptor of the first electron from quinol is the [2Fe-2S] cluster of the Rieske iron-sulfur protein (ISP), which feeds electrons via a bound c-type heme (cytochrome (cyt) c 1 in the bc 1 complex) to a mobile electron carrier protein (cyt c) that then reduces a terminal acceptor (cytochrome oxidase in the mitochondrial chain, an oxidized photochemical reaction center in photosynthetic systems). It is supposed from general principles of quinone chemistry (3) that a semiquinone intermediate is formed at the Q o -site, but this has not been detected. The second electron from quinol is passed to a lower potential chain, containing the low (cyt b L ) and higher (cyt b H ) potential hemes of cyt b, which spans the membrane and delivers electrons to a second quinone processing site on the other side. This is the Q i -site, at which quinone (Q) is reduced to quinol. Because this reaction requires two electrons, the Q o -site has to turn over twice, oxidizing two QH 2 , releasing 4 H ϩ , and delivering two electrons successively to each chain. The Q i -site functions as a two-electron gate, storing the first electron as a bound semiquinone. The two successive one-electron reactions lead to uptake of two H ϩ on complete reduction of quinone. For the mitochondrial complex the overall reaction is shown in Equation 1; in ␣-proteobacteria, cyt c is replaced by cyt c 2 .Here subscripts N and P denote the protochemically negative and positive aqueous phases.The experimental evidence in support of a Q-cycle (1) has been extensively reviewed (see . A modified version of the original Q-cycle (Scheme 1) was proposed to explain apparent anomalies in the pre-steady-state kinetics of the bc 1 complex in chromatophores from the photosynthetic bacteria Rhodobacter sphaeroides and Rhodobacter capsulatus (2,9,12). Although the bacterial systems had provided some of the clearest examples of kinetic effects that were not easily accounted for by earlier Q-cycles (see Ref. 13), similar effects were reported in several studies of the pre-steady-state kinetics in mitochondrial complexes, most notably the extensive work from De Vries (14 -20). The kinetic disparities seemed to be well explained by the modified Q-cycle. The mechanism was highly constrained by the set of reactions, the stoichiometry of components, and by measured physicochemical parameters ...

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The kinetics of light-induced electron transfer in reaction centers (RCs) from the purple photosynthetic bacterium Rhodobacter sphaeroides were studied in the presence of the detergent lauryldimethylamine-N-oxide (LDAO). After the light-induced electron transfer from the primary donor (P) to the acceptor quinone complex, the dark re-reduction of P+ reflects recombination from the reduced acceptor quinones, QA- or QB-. The secondary quinone, QB, which is loosely bound to the RC, determines the rate of this process. Electron transfer to QB slows down the return of the electron to P+, giving rise to a slow phase of the recovery kinetics with time tau P approximately 1 s, whereas charge recombination in RCs lacking QB generates a fast phase with time tau AP approximately 0.1 s. The amount of quinone bound to RC micelles can be reduced by increasing the detergent concentration. The characteristic time of the slow component of P+ dark relaxation, observed at low quinone content per RC micelle (at high detergent concentration), is about 1.2-1.5 s, in sharp contrast to expectations from previous models, according to which the time of the slow component should approach the time of the fast component (about 0.1 s) when the quinone concentration approaches zero. To account for this large discrepancy, a new quantitative approach has been developed to analyze the kinetics of electron transfer in isolated RCs with the following key features: 1) The exchange of quinone between different micelles (RC and detergent micelles) occurs more slowly than electron transfer from QB- to P+; 2) The exchange of quinone between the detergent "phase" and the QB binding site within the same RC micelle is much faster than electron transfer between QA- and P+; 3) The time of the slow component of P+ dark relaxation is determined by (n) > or = 1, the average number of quinones in RC micelles, calculated only for those RC micelles that have at least one quinone per RC (in excess of QA). An analytical function is derived that relates the time of the slow component of P+ relaxation, tau P, and the relative amplitude of the slow phase. This provides a useful means of determining the true equilibrium constant of electron transfer between QA and QB (LAB), and the association equilibrium constant of quinone binding at the QB site (KQ+). We found that LAB = 22 +/- 3 and KQ = 0.6 +/- 0.2 at pH 7.5. The analysis shows that saturation of the QB binding site in detergent-solubilized RCs is difficult to achieve with hydrophobic quinones. This has important implications for the interpretation of apparent dependencies of QB function on environmental parameters (e.g. pH) and on mutational alterations. The model accounts for the effects of detergent and quinone concentration on electron transfer in the acceptor quinone complex, and the conclusions are of general significance for the study of quinone-binding membrane proteins in detergent solutions.

Flash-induced oxygen evolution in the thylakoids of plants and algae exhibits damped oscillations with period four. These are wel described by the S-state model of Kok et al. [Kok, B., Forbush, B. & McGloin, M. (1970) Photochem. Photobiol. 11, with refs. 5-9).Damping of the period four oscillations was explained empirically by misses and double hits (4), the nature ofwhich has never been made fully explicit. The double hit parameter is mainly attributed to a double turnover of the RC induced during the tail of the actinic flash (3, 4).The nature of misses has not been mechanistically defined (see, however, ref. 9). In the analysis of Joliot and Kok (3), misses were suggested to be due either to the fraction of RCs in which a photochemical transition does not occur or to back-reactions that annihilate the effect of the previous flash. Although equal misses for each transition give adequate fitting of the observed oxygen evolution, many authors have suggested that they may be different for each S state (e.g., refs. 3, 9-12) and, indeed, some improvement of the fit is seen. However, these models have been essentially phenomenological in nature.Here we suggest that misses are substantially determined by the fraction of RCs that have either P+ or QA before each flash, due to the reversibility of the electron transfer reactions. With this underlying mechanism, the miss factor becomes a fundamentally informative parameter for the oxygen-evolving process. Calculation of misses from this standpoint, using available values for the equilibrium constants, gives good predictions for flash-induced oxygen evolution. The most important outcome, however, is recognition of two different reaction cycles with different transition probabilities and, consequently, different oxygen yield patterns in a flash series. The relative contributions of the two cycles depend on the initial conditions. This gives rise to an intrinsic, kinetic heterogeneity of PSII activity, which may contribute to the large number of heterogeneities derived from phenomenological analysis (e.g.,

Adrenal cytochrome b(561) (cyt b(561)), a transmembrane protein that shuttles reducing equivalents derived from ascorbate, has two heme centers with distinct spectroscopic signals and reactivity towards ascorbate. The His54/His122 and His88/His161 pairs furnish axial ligands for the hemes, but additional amino acid residues contributing to the heme centers have not been identified. A computational model of human cyt b(561) (Bashtovyy, D., Berczi, A., Asard, H., and Pali, T. (2003) Protoplasma 221, 31-40) predicts that His92 is near the His88/His161 heme and that His110 abuts the His54/His122 heme. We tested these predictions by analyzing the effects of mutations at His92 or His110 on the spectroscopic and functional properties. Wild type cytochrome and mutants with substitutions in other histidine residues or in Asn78 were used for comparison. The largest lineshape changes in the optical absorbance spectrum of the high-potential (b(H)) peak were seen with mutation of His92; the largest changes in the low-potential (b(L)) peak lineshape were observed with mutation of His110. In the EPR spectra, mutation of His92 shifted the position of the g=3.1 signal (b(H)) but not the g=3.7 signal (b(L)). In reductive titrations with ascorbate, mutations in His92 produced the largest increase in the midpoint for the b(H) transition; mutations in His110 produced the largest decreases in DeltaA(561) for the b(L) transition. These results indicate that His92 can be considered part of the b(H) heme center, and His110 part of the b(L) heme center, in adrenal cyt b(561).