Electron transfer through the isolated mitochondrial cytochrome b-c1 complex.

(1) A kinetic analysis of electron donation into and through the cytochrome b-c1 complex isolated from bovine heart mitochondria has been undertaken, using trimethoquinol as the donor. (2) Rate constants of two routes of redox equilibration with quinols have been defined by kinetic measurements and with the use of the inhibitors antimycin A and myxothiazol. (3) A model of electron transfer based upon the original Q-cycle formulation is presented to explain these and related results.

The effect of pH on redox titrations of haem a in cyanide-liganded cytochrome-c oxidase: experimental and modelling studies.

Isolated cytochrome-c oxidase ligated with cyanide was titrated by Flash-Induced chemical photoREduction (FIRE) (Moody, A.J. and Rich, P.R. (1988) EBEC Short Rep. 5, 69) using cytochrome c as a redox indicator. Haem a is found to titrate in a complex manner consistent with its interacting anticooperatively with at least two other components. We assign CuB as the major interactant at neutral pH, and CuA as the minor interactant. In the pH range 7.0-8.1 the strength of the interaction with CuB is found to decrease with increasing pH, while the interaction with CuA remains essentially constant. The decrease in the interaction with CuB appears to continue above pH 8.1 such that at pH 9.2 the titration curve for haem a is only slightly distorted from an 'n = 1' shape, although it is not possible from the titration data to assess the relative contributions of CuB and CuA to the total interaction observed at pH values greater than 8.1. Haem a and CuB show similar pH-dependence and, to account for this, we present a model in which the oxidoreductions of both haem a and CuB are linked to the (de)protonation of a common acid/base group. The model predicts a pH-dependent indirect cooperative interaction between haem a and CuB in addition to the direct anticooperative interaction, thereby explaining the observed pH-dependence of the redox interaction between haem a and CuB.

Light-activated proton-motive force generation in lipid vesicles containing cytochrome b-c1 complex and bacterial reaction centres.

(1) Purified bovine heart mitochondrial cytochrome b-c1 complex (ubiquinone-cytochrome c oxidoreductase) and photosynthetic reaction centres isolated from Rhodopseudomonas sphaeroides strain R-26 have been incorporated into lipid vesicles. In the presence of cytochrome c and ubiquinone-2, light activation caused a cyclic electron transfer involving both components. (2) Since cytochrome c is added outside the vesicles, it is both reduced by the cytochrome b-c1 complex and oxidised by the reaction centre on the outside of the vesicles. Ubiquinone-2, however, is reduced by the reaction centres at a site in contact with the inside of the vesicles, but the reduced form, ubiquinol-2, is oxidised by the cytochrome b-c1 complex at a site in contact with the outer aqueous phase. (3) In the presence of valinomycin plus K+, initiation of cyclic electron flow causes protons to move from inside the vesicles to the outer medium and the H +/2e- ratio was calculated to be close to 4.

Proton uptake by cytochrome c oxidase on reduction and on ligand binding.

On reduction, cytochrome oxidase was found to take up 2.4 +/- 0.1 protons in the pH range 7.2-8.5, of which 2 are associated with the binuclear centre, and the remaining fractional proton with haem a/CuA. Ligation to oxidised cytochrome oxidase of the azide, formate, fluoride or cyanide anions is accompanied by uptake of one proton. In the case of the reduced enzyme, no protonation changes are observed on binding O2 (Hallén S. and Nilsson T. (1992) Biochemistry 31, 11853-11859) or CO. Cyanide binding to reduced oxidase is, in contrast, still accompanied by uptake of a proton. These findings are discussed in terms of our previously-published proposal for the ligand chemistry of the binuclear site. The results overall suggest a principle of electroneutrality of redox and ligand state changes of the binuclear centre, with charge compensations provided only by protonation reactions.

The functional catalytic unit involved in proton pumping by rat liver cytochrome-c reductase and by cytochrome-c oxidase.

The effect of partial inhibition on the protonmotive stoichiometry of cytochrome-c reductase and cytochrome-c oxidase in intact rat liver mitochondria was examined using myxothiazol and cyanide as inhibitors, respectively. No decrease in the stoichiometry of either enzyme was found. It is shown that this result is consistent with the individual electron transfer units in each case being fully coupled to proton translocation but not with pairs of electron transfer units working in concert in dimers.

EPR studies of higher plant mitochondria. II. Center S-3 of succinate dehydrogenase and its relation to alternative respiratory oxidations.

1. An electron paramagnetic resonance study of the high potential iron sulfur (HiPIP-type) Center S-3 of higher plant mitochondria is described. This center is the major HiPIP-type center associated with plant mitochondria and it displays physical properties which are similar to its mammalian counterpart. It has a pH-independent midpoint potential of +65 +/- 10 mV between pH 6.0 and 8.5. 2. The behavior of Center S-3 in a variety of steady-state conditions suggests that it is of physiological significance in electron transport. Furthermore, it can be shown that the alternative oxidase, which is present in many higher plant mitochondria, tends to keep this center oxidized in the presence of succinate and cyanide. This indicates that the alternative oxidation site is on the electron-donating side of the Center S-3. 3. Salicylhydroxamic acid, an inhibitor of the alternative pathway, does not affect the midpoint potential, signal size or shape, or temperature and power saturation profiles of Center S-3, suggesting that direct autoxidation of this center cannot account for alternative oxidase activity. This is further confirmed by the finding that the presence of succinate dehydrogenase is not necessary for alternative oxidase activity with NADH as respiratory substrate in submitochondrial particles.

An EPR analysis of cyanide-resistant mitochondria isolated from the mutant poky strain of Neurospora crassa.

An analysis of the paramagnetic components present in mitochondria isolated from the poky mutant of Neurospora crassa is described. The study was undertaken with a view to shedding light on the nature of the cyanide- and antimycin A-resistant alternative terminal oxidase which is present in these preparations. Of the ferredoxin-type iron-sulfure centers, only Centers S-1 and S-2 of succinate dehydrogenase could be detected in significant quantities. Paramagnetic centers attributable to Site I were virtually absent. In the oxidized state, at least two 'high potential iron sulfur' centers could be distinguished and these were attributed to Center S-3 of succinate dehydrogenase and a second component analogous to that found in mammalian systems. Much of the Center S-3 signal was in a highly distorted state which was apparently dependent upon the presence of an accompanying free radical species. At lower field positions, a succinate-reducible signal peaking around g = 3.15 was found. This signal is caused by a low spin heme species, presumably the cytochrome c which is the only major cytochrome in these mitochondria. At even lower field positions, signals attributable to iron in a field of low symmetry at g = 4.3 and multiple high spin heme species around g = 6, could be distinguished. The effects of salicylhydroxamic acid, an inhibitor of the alternative oxidase, were tested on these components. Effects could be seen on at least one high spin heme component and also partially upon the distorted Center S-3 signal converting part of it to a signal indistinguishable from center S-3. Some increase in the g = 4.3 iron signal was also noted. No effects of the inhibitor on the ferredoxin-type centers were detected.

The kinetics and thermodynamics of the reduction of cytochrome c by substituted p-benzoquinols in solution.

1. The mechanisms by which p-benzoquinol and its derivatives reduce cytochrome c in solution have been investigated. 2. The two major reductants are the species QH- (anionic quinol) and Q.- (anionic semiquinone). A minor route of electron transfer from the fully protonated QH2 species can also occur. 3. The relative contributions of these routes to the overall reduction rate are governed by pH, ionic strength and relative reactant concentrations. 4. For a series of substituted p-benzoquinols, the forward rate constant, k1, of the anionic quinol-mediatd reaction is related to the midpoint potential of the QH-/QH. couple involved in the rate-limiting step, as predicted by the theory of Marcus for outer-sphere electron transfer reactions in a bimolecular collision process. 5. A mechanism for the biological quinol oxidation reactions in mitochondria and chloroplasts is proposed based upon the findings with these reactions in solution.

The redox potentials of the b-type cytochromes of higher plant chloroplasts.

1. In fresh chloroplasts,three b-type cytochromes exist. These are b-559HP (lambda max, 559 nm; Em at pH 7, +370 mV; pH-independent Em), b-559LP (lambda max, 559 nm; Em at pH 7, +20 mV; pH-independent Em) and b-563 (lambda max, 563 nm; Em at pH 7, -110 mV; pH-independent Em), b-559HP may be converted to a lower potential form (lambda max, 559 nm; Em at pH 7, +110 mV; pH-independent Em). 2. In catalytically active b-f particle preparations, three cytochromes exist. These are cytochrome f (lambda max, 554 nm; Em at pH 7, +375 mV, pK on oxidised cytochrome at pH 9), b-563 (lambda max, 563 nm; Em at pH 7, -90 mV, small pH-dependence of Em) and a b-559 species (lambda max, 559 nm, Em at pH 7, +85 mV; pH-independent Em). 3. A positive method of demonstration and estimation of b-559LP in fresh chloroplasts is described which involves the use of menadiol as a selective reductant of b-559LP.

The reactions of hydrogen peroxide with bovine cytochrome c oxidase.

Oxidised cytochrome c oxidase is known to react with two molecules of hydrogen peroxide to form consecutively 607 nm 'Peroxy' and 580-nm 'Ferryl' species. These are widely used as model compounds for the equivalent P and F intermediates of the catalytic cycle. However, kinetic analysis of the reaction with H(2)O(2) in the pH range 6.0-9.0 reveals a more complex situation. In particular, as the pH is lowered, a 580-nm compound can be formed by reaction with a single H(2)O(2). This species, termed F(&z.rad;), is spectrally similar, but not identical, to F. The reactions are equivalent to those previously reported for the bo type quinol oxidase from Escherichia coli (T. Brittain, R.H. Little, C. Greenwood, N.J. Watmough, FEBS Lett. 399 (1996) 21-25) where it was proposed that F(&z.rad;) is produced directly from P. However, in the bovine oxidase F(&z.rad;) does not appear in samples of the 607-nm form, P(M), produced by CO/O(2) treatment, even at low pH, although this form is shown to be identical to the H(2)O(2)-derived P state, P(H), on the basis of spectral characteristics and kinetics of reaction with H(2)O(2). Furthermore, lowering the pH of a sample of P(M) or P(H) generated at high pH results in F(&z.rad;) formation only on a minutes time scale. It is concluded that P and F(&z.rad;) are not in a rapid, pH-dependent equilibrium, but instead are formed by distinct pathways and cannot interconvert in a simple manner, and that the crucial difference between them lies in their patterns of protonation.

Electron conduction between b cytochromes of the mitochondrial respiratory chain in the presence of antimycin plus myxothiazol.

The b haems of the bc1 complex of bovine heart mitochondria were poised with succinate and fumarate so that only the high-potential haem (b-562) was reduced, and then isolated from further redox exchange with the ubiquinone pool by adding antimycin and myxothiazol. A transmembrane electric potential difference was then developed, either by electron flow from [Ru(NH3)6]Cl2 to oxygen or by ATP hydrolysis. The small difference spectrum, caused by the electric field, indicated 32-55% oxidation of b-562 with concomitant reduction of b-566. No lag greater than 0.1 s was detectable between the initiation of respiration and the development of the difference spectrum, thus providing a direct demonstration of (fairly) rapid electron transfer between the b haems.

Protonation states of the catalytic intermediates of cytochrome c oxidase.

Protonation changes accompanying conversion of oxidised (O state) cytochrome c oxidase to the 2-electron-reduced P state, and 3-electron-reduced F state at pH 8.0 have been measured. It was found that 2 and 3 protons, respectively, were taken up. The fourth proton required for the reduction of O2 to H2O must therefore be consumed in the remaining F----O portion of the catalytic cycle.

The topology of CuA in relation to the other metal centres in cytochrome-c oxidase of Saccharomyces cerevisiae as determined by analysis of second-site reversions.

Second-site revertants were selected from a respiratory-deficient mutant carrying the mutation D369N located in a loop between helices IX and X close to H376 and H378, the proposed ligands of haem a3 and haem a, respectively. A reversion was observed in subunit II, in the vicinity of the CuA ligands. This same reversion compensates the subunit I deficiency mutation, S140L, assumed to be near H62, the second putative histidine ligand to haem a. These data enable us to propose a three-dimensional topology in which CuA in subunit II is located on top of the Positive-side of subunit I and in proximity to all three of its metal centres.

Rates of cyanide binding to the catalytic intermediates of mammalian cytochrome c oxidase, and the effects of cytochrome c and poly(L-lysine).

Rate constants of cyanide binding to 'fast' oxidase have been measured in the fully-oxidised (O), peroxy (P) and ferryl (F) states at pH 8.0. Values of 2.2, 8 and 10 M-1 s-1, respectively, were obtained. Thus, none of these states appears to exhibit a rate that would identify it as the species responsible for the extremely rapid cyanide binding observed during turnover. On the other hand, with 'oxidised' enzyme as prepared, containing a very small fraction of one-electron-reduced (E state) oxidase, a corresponding fraction of enzyme exhibited spectral changes consistent with cyanide binding with a rate constant in excess of 10(4) M-1 s-1. Evidence is presented suggesting that mediation of electron transfer from one-electron-reduced, cyanide-liganded enzyme to free, ferric oxidase, rather than a global protein conformational change of the enzyme, is responsible for the greatly enhanced cyanide binding rates seen in the presence of cytochrome c or poly(L-lysine). Inter-oxidase electron exchange in 'oxidised' enzyme can result in a complicated dependence of the binding rate on cyanide concentration. We have demonstrated that this may give rise to a saturation of the rate of cyanide binding.

Protonation reactions in relation to the coupling mechanism of bovine cytochrome c oxidase.

Identification of the locations of protonatable sites in cytochrome c oxidase that are influenced by reactions in the binuclear centre is critical to assessment of proposed coupling mechanisms, and to controversies on where the pumping steps occur. One such protonation site is that which governs interconversion of the isoelectronic 607 nm 'P(M)' and 580 nm 'F' forms of the two-electron-reduced oxygen intermediate. Low pH favours protonation of a site that is close to an electron paramagnetic resonance (EPR)-silent radical species in P(M), and this induces a partial electronic redistribution to form an EPR-detectable tryptophan radical in F. A further protonatable group that must be close to the binuclear centre has been detected in bacterial oxidases by Fourier transform infrared spectroscopy from pH-dependent changes in the haem-bound CO vibration frequency at low temperatures. However, in bovine cytochrome c oxidase under similar conditions of measurement, haem-bound CO remains predominantly in a single 1963 cm(-1) form between pH 6.5 and 8.5, indicating that this group is not present. Lack of pH dependence extends to the protein region of the CO photolysis spectra and suggests that both the reduced and the reduced/CO states do not have titratable groups that affect the binuclear centre strongly in the pH range 6.5-8.5. This includes the conserved glutamic acid residue E242 whose pK appears to be above 8.5 even in the fully oxidised enzyme. The results are discussed in relation to recent ideas on coupling mechanism.

Characterisation of 'fast' and 'slow' forms of bovine heart cytochrome-c oxidase.

We have prepared cytochrome-c oxidase from bovine heart (using a modification of the method of Kuboyama et al. (1972) J. Biol. Chem. 247, 6375-6383) which binds cyanide rapidly, shows no kinetic distinction between the two haems on reduction by dithionite, has a Soret absorption maximum above 424 nm, and has a negligible 'g' = 12' EPR signal. On incubation at pH 6.5 this 'fast' oxidase reverts to the 'slow' ('resting') form characterised by slow cyanide binding, slow reduction of haem a3 by dithionite, a blue-shifted Soret maximum and a large 'g' = 12' signal. Incubation of 'fast' oxidase with formate produces a form of the enzyme with properties almost identical to those of 'slow' oxidase. The kinetics of formate binding to 'fast' oxidase are found to be biphasic, revealing the presence of at least two 'fast' subpopulations in our preparations. Evidence is presented that there is an equilibrium mixture of high-spin and low-spin forms of haem a3 in both 'fast' subpopulations at room temperature. Incubation of 'fast' oxidase with chloride or bromide at pH 6.5 produces forms of oxidase with much lower rates of cyanide binding. Our working hypothesis is that formate mimics a binuclear centre ligand which is present in the 'slow' form of cytochrome oxidase. Although we show that chloride and bromide can also be ligands of the binuclear centre, possibly onto CuB, we can rule out either of these being the ligand present in the 'slow' enzyme. We will argue that the 'fast' and 'slow' forms of oxidase are equivalent to the 'pulsed' and 'resting' forms of oxidase, respectively.

Exchange integral for a variety of tetranuclear ferredoxins.

The temperature dependence of EPR spectra of oxidized [4Fe-4S](-1,-2) ferredoxins (previously designated HiPIP) and a reduced [4Fe-4S](-2,-3) ferredoxin have been analyzed so as to determine the energy of a low-lying excited electronic state. The values obtained were: Center S-3 from beef heart, 44 cm-1; Center S-3 from mung bean, 53 cm-1; the [4Fe-4S](-1,-2) ferredoxin from Thermus thermophilus, 78 cm-1; Center N-2 of NADH ubiquinone reductase, 83 cm-1. Increasing axial distortion in the EPR spectra of the [4Fe-4S](-1,-2), ferrodoxins was associated with higher energy differences. Center N-2, a [Fe-4S](-2,-3) iron-sulfur cluster does not fit this relationship.

Studies on the molecular organization of cytochromes P-450 and b5 in the microsomal membrane.

1. The relative orientations of the heme groups of cytochromes P-450 and b5 in the microsomal membrane have been studied by the technique of electron paramagnetic resonance. The results show that the heme plane of cytochrome P-450 lies in the same plane as the membrane surface, whereas the cytochrome b5 heme plane has a random orientation. 2. No significant broadening or change in relaxation properties of the gz component of low spin cytochrome P-450 occurred when cytochrome b5 was reduced by redox poising. It is concluded that there is little or no paramagnetic coupling between the heme groups of the two species. 3. The results favor a model in which no tight complex between cytochromes P-450 and b5 is present, the species being independent and interacting only by random molecular collisions or via other intermediate species.

Inhibitor effects on redox-linked protonations of the b haems of the mitochondrial bc1 complex.

The effects of pH and inhibitors on the spectra and redox properties of the haems b of the bc1 complex of beef heart submitochondrial particles were investigated. The major findings were: (1) both haems have a weakly redox-linked protonatable group with pKox and pKred of around 6 and 8; (2) at pH values above 7, haem bH becomes heterogeneous in its redox behaviour. This heterogeneity is removed by the Qi site inhibitors antimycin A, funiculosin and HQNO, but not by the Qo site inhibitors myxothiazol or stigmatellin; (3) of all inhibitors tested only funiculosin had a large effect on the Em/pH profile of either haem b. In all cases where definite effects were found, the haem most affected was that thought to be closest to the site of inhibitor binding; (4) spectral shifts of haem groups caused by inhibitor binding were usually, but not always, of the haem group closest to the binding site; (5) titrations with succinate/fumarate were in reasonable agreement with redox-mediated data provided that strict anaerobiosis was maintained. Apparent large shifts of haem midpoint potentials with antimycin A and myxothiazol could be produced in aerobic succinate/fumarate titrations in the presence of cyanide, as already reported in the literature, but these were artefactual; (6) the heterogeneous haem bH titration behaviour can be simulated with a model similar to that proposed by Salerno et al. (J. Biol. Chem. (1989) 264, 15398-15403) in which there is redox interaction between haem bH and ubiquinone species bound at the Qi site. Simulations closely fit both the haem bH data and known semiquinone data only if it is assumed that semiquinone bound to oxidised haem bH is EPR-silent.

EPR studies of higher plant mitochondria. I Ubisemiquinone and its relation to alternative respiratory oxidations.

An EPR investigation of the region of the higher plant respiratory chain involving ubiquinone and Center S-3 of succinate dehydrogenase is reported. At temperatures close to those of liquid helium, first derivative spectra corresponding to Center S-3 (gmax = 2.017) and a signal split around g = 2.00 (major features of peaks and troughs at g values of 2.045, 2.03, 1.985, 1.97 and 1.96) were observed in mung bean (Phaseolus aureus), Arum maculatum spadix, Sauromatum guttatum spadix and tulip bulb (Tulipa gesnerana) mitochondria. The split signal was small or absent in potato tuber and Symplocarpus foetidus spadix mitochondria. The redox behavior of these signals in mung bean mitochondria in a variety of respiratory steady-state conditions suggested that the components giving rise to them were an integral part of the respiratory chain and were located on the substrate side of coupling Site II. The split signal could be removed by addition of hydroxamic acids in all tissues tested, although the Ks of this effect was an order of magnitude higher than the Ki of inhibition of the alternative respiratory pathway in mung bean and Sauromatum guttatum spadix mitochondria. The results are discussed in relation to the current ideas on the ordering of components in the region around the classical Site II of the respiratory chain and in relation to the location of the alternative respiratory oxidase pathway of higher plants.