Control of proteoliposomal cytochrome c oxidase: the partial reactions

The steady-state spectroscopic behaviour and the turnover of cytochrome c oxidase incorporated into proteoliposomes have been investigated as functions of membrane potential and pH gradient. The respiration rate is almost linearly dependent on [cytochrome c2+] at high flux, but while the cytochrome a redox state is always dependent on the [cytochrome c2+] steady state, it reaches a maximum reduction level less than 100% in each case. The maximal aerobic steady-state reduction level of cytochrome a is highest in the presence of valinomycin and lowest in the presence of nigericin. The proportion of [cytochrome c2+] required to achieve 50% of maximal reduction of cytochrome a varies with the added ionophores; the apparent redox potential of cytochrome a is most positive in the fully decontrolled system (plus valinomycin and nigericin). At low levels of cytochrome a reduction, the rate of respiration is no longer a linear function of [cytochrome c2+], but is dependent upon the redox state of both cytochromes a and c. That is, proteoliposomal oxidase does not follow Smith-Conrad kinetics at low cytochrome c reduction levels, especially in the controlled states. The control of cytochrome oxidase turnover by delta pH and by delta psi can be explained either by an allosteric model or by a model with reversed electron transfer between the binuclear centre and cytochrome a. Other evidence suggests that the reversed electron transfer model may be the correct one.     

The dioxygen cycle. Spectral, kinetic, and thermodynamic characteristics of ferryl and peroxy intermediates observed by reversal of the cytochrome oxidase reaction.

The catalytic mechanism of O2 reduction by cytochrome oxidase was studied in isolated mitochondria and mitoplasts by partial reversal of the reaction. At a high redox potential (Eh) of cytochrome c, high pH, and a high electrochemical proton gradient (delta mu H+) across the inner mitochondrial membrane, the initial ferriccupric state (O) of the oxidized enzyme's bimetallic oxygen reaction center is converted to ferryl (F) and peroxy (P) intermediates, the optical spectroscopic properties of which are reported in detail. This is associated with reversed electron transfer from the bimetallic center to ferricytochrome c. The kinetics of reduction of ferricytochrome c by the reversed electron transfer process are compared with the kinetics of formation of F and P. The results are consistent with transfer of one electron from the ferric-cupric bimetallic center (O) to cytochrome c, yielding the F intermediate, followed by transfer of one electron from the latter to cytochrome c, yielding the P state. In the absence of an effective redox buffer, poising cytochrome c highly oxidized, these primary events are immediately followed by reoxidation of cytochrome c, which is ascribed to forward electron transfer to enzyme molecules still in the O state. This forward reaction also results in accumulation of the P intermediate. Kinetic stimulations of the data predict equilibrium constants for the reversed electron transfer steps, and Em,7 values of approximately 1.1 and 1.2 V may be calculated for the F/O and P/F redox couples, respectively, at delta mu H+ and delta psi equal to zero. Taken together with previously measured Em,7 values, these data indicate that it is the two-electron reduction of bound dioxygen to bound peroxide that is responsible for the irreversibility of the catalytic dioxygen cycle of cell respiration.     

Effect of membrane potential and pH gradient on electron transfer in cytochrome oxidase

Steady-state spectra of cytochrome oxidase in phospholipid vesicles were obtained by using hexaammineruthenium(II) and ascorbate as reductants. Cytochrome a was up to 80% reduced in the steady state in coupled vesicles. Upon addition of nigericin or acetate, which decrease delta pH, resulting in an increase in delta psi, cytochrome a became more oxidized in the steady state with no change in the rate of respiration. On the other hand, uncouplers or valinomycin plus nigericin, which lower both delta psi and delta pH, stimulated respiration 2-8-fold and also lowered the steady-state level of reduction of cytochrome a. These experiments indicate that electron transfer between cytochromes a and a 3 is sensitive primarily to the pH gradient. Studies with the reconstituted and the soluble enzyme at various pH values indicated that the pH on the matrix side of the membrane, rather than delta pH, controlled the steady-state level of reduced cytochrome a. Hexaammineruthenium(II) substituted for cytochrome c in measurements of proton pumping by cytochrome oxidase. Dicyclohexylcarbodiimide, which eliminated proton pumping by cytochrome oxidase, decreased the effect of ionophores on the steady-state level of reduced cytochrome a.     

Independent control of respiration in cytochrome c oxidase vesicles by pH and electrical gradients

The effects of altering the pH and electrical components of the membrane potential on the visible spectra and oxygen consumption rates of cytochrome oxidase vesicles were examined during steady-state respiration using cytochrome c as the substrate. Heme a was found to be 30-55% reduced in the presence of a membrane potential, becoming more reduced when the electrical gradient (delta psi) was abolished by valinomycin and more oxidized when the pH gradient (delta pH) was abolished by nigericin, with little increase (1.2-1.8-fold) in the rates of oxygen consumption in either case. When both gradients were eliminated, heme a reduction was close to initial levels, and activity was stimulated up to 8-fold. The magnitude of the changes in heme a reduction levels upon elimination of a gradient component was shown to be positively correlated with the magnitude of the respiratory control ratio of the vesicle preparation. Kinetic analysis of the dependence of oxidase activity on cytochrome c concentration indicated that changes in the Michaelis constant of the enzyme for its substrate are not a major factor in regulation by either delta pH or delta psi. These results suggest a dual mechanism for respiratory control in cytochrome oxidase vesicles under steady-state conditions, in which the electrical gradient predominantly affects electron transfer from cytochrome c to heme a, possibly by altering the reduction potential of heme a, while the pH gradient affects electron transfer from heme a (CuA) to heme a3 (CuB), possibly by a conformationally mediated change in the reduction potential of heme a3 or in the kinetics of the electron-transfer process.     

Control of proteoliposomal cytochrome c oxidase: the overall reaction

The control of cytochrome c oxidase incorporated into proteoliposomes has been investigated as a function of membrane potential (delta psi) and pH gradient (delta pH). The oxidase generates a pH gradient (alkaline inside) and a membrane potential (negative inside) when respiring on external cytochrome c. Low levels of valinomycin collapse delta psi and increase delta pH; the respiration rate decreases. High levels of valinomycin, however, decrease delta pH as valinomycin can also act as a protonophore. Nigericin (in the absence of valinomycin) increases delta psi and collapses delta pH; the respiration rate increases. On a millivolt equivalent basis delta pH is a more effective inhibitor of activity than is delta psi. In the absence of any ionophores the cytochrome oxidase proteoliposomes enter a steady state, in which there are both delta pH and delta psi components of control. Present and previous data suggest that the respiration rate responds in a linear way ("ohmically") to increasing delta pH but in a nonlinear way to delta psi ("non-ohmically"). High levels of both delta psi and delta pH do not completely inhibit turnover (maximal respiratory control values lie between 6 and 10). The controlled steady state involves the electrophoretic entry and electroneutral exit of K+ from the vesicles. A model is presented in which the enzyme responds to both delta pH and delta psi components of the proton-motive force, but is more sensitive to delta pH than to delta psi at an equivalent delta mu H+. The steady state of the proteoliposome system can be represented for any set of permeabilities and enzyme activity levels using the computer simulation programme Stella.     

Thermodynamic control of electron flux through mitochondrial cytochrome bc1 complex.

The redox states of exogenously added ubiquinone-2 and cytochrome c, and the protonmotive force (delta p) of rat liver mitochondria were measured as the respiration rate was titrated with the uncoupler carbonyl cyanide p-trifluoromethoxyphenyl-hydrazone. The force ratio delta Eh/delta p across the bc1 complex was close to 1:1 in State 4, indicating an H+/e- stoichiometry of 1:1 for the cytochrome bc1 complex, excluding protons moved by pool ubiquinone. Assuming a constant stoichiometry the rate of electron transport increased linearly with the disequilibrium (delta Eh - delta p) across the complex.     

Characterization of a new membrane-bound cytochrome c of Rhodopseudomonas capsulata

A cytochrome c (cyt. c) was solubilized with Triton-X-100 and co-purified with cytochrome c oxidase from membranes of chemotrophically grown cells of Rhodopseudomonas capsulata. Cyt. c and cytochrome oxidase were separated on Sephadex G-50 columns. Antibodies against cytochrome c2 from the same bacterium did not cross react with the membrane-bound cyt. c. The IEP of the membrane-bound cyt. c was found to be pH 8.2, the midpoint potential was 234 +/- 11 mV at pH 7.0. This cyt. c binds CO. The native cyt. c is a dimer with an apparent Mr of 25000 containing 2 mol heme per mol dimer, which is believed to function as an electron donor for the high-potential cytochrome c oxidase.     

Electron transfer reactions in the NADPH oxidase system of neutrophils--involvement of an NADPH-cytochrome c reductase in the oxidase system.

Membrane-bound NADPH oxidase of pig blood neutrophils was solubilized with heptylthioglucoside in a high yield. The solubilized preparation from myristate-stimulated cells (sample S) showed high O2- generating activity, and the preparation from resting cells (sample R) had no activity, but the two samples had equal amounts of flavins and cytochrome b-558 (cyt b-558). The electron transfer reactions to exogenous cytochrome c (cyt c) or cyt b-558 in samples S and R were examined. Under anaerobic conditions, NADPH-dependent cyt c reductase activity appeared higher in sample S than in sample R, and the addition of FMN and FAD greatly enhanced the reductase activity of sample S, but not that of sample R. No marked difference between the reductase activities of samples S and R was seen with NADH. Photoreduction of the NADPH oxidase system was examined in the absence of NADPH under anaerobic conditions by monitoring the reduction rates of exogenous cyt c using a flashlight with cut-off filters between 400 and 500 nm. Cyt c reduction was much higher in sample S than in sample R on photoexcitation at about 450 nm. Photoreduction was carried out with a band-pass filter for selective irradiation at 450 nm. Marked reduction of exogenous cyt c was observed only in sample S: the small reduction of cyt c by sample R was independent of the light wavelength and was equal to the blank level. In contrast, no difference in the reduction of cyt b-558 by the two samples was found by either NADPH or photoreduction. Under aerobic conditions, no direct reduction of either cyt c or cyt b-558 was observed. These results suggest that an NADPH-cyt c reductase (a membrane-bound flavoprotein) is involved in the NADPH oxidase system of stimulated neutrophils.     

New control of mitochondrial membrane potential and ROS formation--a hypothesis

A new control of mitochondrial membrane potential delta(psi)m and formation of reactive oxygen species (ROS) is presented, based on allosteric ATP-inhibition of cytochrome c oxidase at high intramitochondrial ATP/ADP ratios. Since the rate of ATP synthesis by the ATP synthase is already maximal at low membrane potentials (100-120 mV), the ATP/ADP ratio will also be maximal at this delta(psi)m (at constant rate of ATP consumption). Therefore the control of respiration by the ATP/ADP-ratio keeps delta(psi)m low. In contrast, the known 'respiratory control' leads to an inhibition of respiration only at high delta(psi)m values (150-200 mV) which cause ROS formation. ATP-inhibition of cytochrome c oxidase is switched on and off by reversible phosphorylation (via cAMP and calcium, respectively). We propose that 'stress hormones' which increase intracellular [Ca2+] also increase delta(psi)m and ROS formation, which promote degenerative diseases and accelerate aging.     

Hexaammineruthenium as an electron donor to mitochondrial cytochrome oxidase: membrane potential generation in the absence of cytochrome c

Cytochrome c oxidase can generate membrane potential in the absence of cytochrome c (e.g., in cytochrome c-deficient mitochondria or in proteoliposomes) with hexaammineruthenium as an artificial electron donor. Of several other redox mediators tested, phenazine methosulfate was found to be an efficient artificial substrate for membrane energization by cytochrome oxidase, whereas TMPD, DAD, DCPIP or ferrocyanide are virtually ineffective. The ability of Ru(NH3)6(2+) and phenazine methosulfate to support the generation of delta psi by cytochrome c-oxidase correlates with their effectiveness as electron donors to cytochrome a in the cyanide-inhibited membrane-bound enzyme.     

Protonmotive activity of cytochrome c oxidase: control of oxidoreduction of the heme centers by the protonmotive force in the reconstituted beef heart enzyme

This paper contributes to the characterization of partial steps of electron and proton transfer in mitochondrial cytochrome c oxidase with respect to their membrane arrangement and involvement in energy-linked protonmotive activity. It is shown that delta psi controls electron flow from cytochrome c to heme a is consistent with the view that the latter center is buried in the membrane in a central position. The pressure exerted by delta psi on oxidation of heme alpha 3 by O2 indicates also that this center is buried in the membrane at some distance from the inner side and is consistent with observations showing that protons consumed in the reduction of O2 to H2O derive from the inner space. Electron flow from heme alpha to heme alpha 3 is shown to be specifically controlled by delta pH and in particular by the pH of the inner phase. Analysis of the effect of DCCD treatment of oxidase vesicles reveals that concentrations of this reagent which result in selective modification of subunit III (Prochaska et al., 1981) produce inhibition of redox-linked proton release. Higher concentrations of DCCD which result also in modification of subunits II and IV (Prochaska et al., 1981) cause inhibition of the pH-dependent electron-transfer step from heme alpha to heme alpha 3.     

Function of the cytochrome bc1-aa3 branch of the respiratory network in mycobacteria and network adaptation occurring in response to its disruption

The aerobic electron transport chain in Mycobacterium smegmatis can terminate in one of three possible terminal oxidase complexes. The structure and function of the electron transport pathway leading from the menaquinol-menaquinone pool to the cytochrome bc1 complex and terminating in the aa3-type cytochrome c oxidase was characterized. M. smegmatis strains with mutations in the bc1 complex and in subunit II of cyctochome c oxidase were found to be profoundly growth impaired, confirming the importance of this respiratory pathway for mycobacterial growth under aerobic conditions. Disruption of this pathway resulted in an adaptation of the respiratory network that is characterized by a marked up-regulation of cydAB, which encodes the bioenergetically less efficient and microaerobically induced cytochrome bd-type menaquinol oxidase that is required for the growth of M. smegmatis under O2-limiting conditions. Further insights into the adaptation of this organism to rerouting of the electron flux through the branch terminating in the bd-type oxidase were revealed by expression profiling of the bc1-deficient mutant strain using a partial-genome microarray of M. smegmatis that is enriched in essential genes. Although the expression profile was indicative of an increase in the reduced state of the respiratory chain, blockage of the bc1-aa3 pathway did not induce the sentinel genes of M. smegmatis that are induced by oxygen starvation and are regulated by the DosR two-component regulator.     

Control of electron transfer in the cytochrome system of mitochondria by pH, transmembrane pH gradient and electrical potential. The cytochromes b-c segment.

1. A study is presented of the effects of pH, transmembrane pH gradient and electrical potential on oxidoreductions of b and c cytochromes in ox heart mitochondria and 'inside-out' submitochondrial particles. 2. Kinetic analysis shows that, in mitochondria at neutral pH, there is a restraint on the aerobic oxidation of cytochrome b566 with respect to cytochrome b562. Valinomycin plus K+ accelerates cytochrome b566 oxidation and retards net oxidation of cytochrome b562. At alkaline pH the rate of cytochrome b566 oxidation approaches that of cytochrome b562 and the effects of valinomycin on b cytochromes are impaired. 3. At slightly acidic pH, oxygenation of antimycin-supplemented mitochondria causes rapid reduction of cytochrome b566 and small delayed reduction of cytochrome b562. Valinomycin or a pH increase in the medium promote reduction of cytochrome b562 and decrease net reduction of cytochrome b566. 4. Addition of valinomycin to mitochondria and submitochondrial particles in the respiring steady state causes, at pH values around neutrality, preferential oxidation of cytochrome b566 with respect to cytochrome b562. The differential effect of valinomycin on oxidation of cytochromes b566 and b562 is enhanced by substitution of 1H2O of the medium with 2H2O and tends to disappear as the pH of the medium is raised to alkaline values. 5. Nigericin addition in the aerobic steady state causes, both in mitochondria and submitochondrial particles, preferential oxidation of cytochrome b562 with respect to cytochrome b566. This is accompanied by c cytochrome oxidation in mitochondria but c cytochrome reduction in submitochondrial particles. 6. In mitochondria as well as in submitochondrial particles, the aerobic transmembrane potential (delta psi) does not change by raising the pH of the external medium from neutrality to alkalinity. The transmembrane pH gradient (delta pH) on the other hand, decrease slightly. 7. The results presented provide evidence that the delta psi component of the aerobic delta microH+ (the sum of the proton chemical and electrical activities) exerts a pH-dependent constraint on forward electron flow from cytochrome b566 to cytochrome b562. This effect is explained as a consequence of anisotropic location of cytochromes b566 and b562 in the membrane and the pH-dependence of the redox function of these cytochromes. Transmembrane delta pH, on the other hand, exerts control on electron flow from cytochrome b562 to c cytochromes.     

Membrane potentials in reconstituted cytochrome c oxidase proteoliposomes determined by butyltriphenyl phosphonium cation distribution

Equilibration of the butyltriphenyl phosphonium (BTPP+) cation into cytochrome c oxidase reconstituted proteoliposomes was measured potentiometrically. The maximum membrane potential (delta psi) generated by oxidase activity was estimated to lie between -65 and -90 mV, vesicle interior negative, when internal BTPP+ binding is taken into account. Formation of delta psi was completely prevented by valinomycin and carbonyl-cyanide-p-trifluoromethoxyphenylhydrazone but only 10% inhibited by levels of N',N'-dicyclohexylcarbodiimide that abolish proton pumping by the oxidase. delta psi is thus maintained by at least one charge transfer process that does not involve proton movement. A nonlinear relationship was obtained between oxidase activity and steady-state delta psi. The value of delta psi estimated by BTPP+ distribution was lower than that calculated using the optical probes safranine and a carbocyanine dye. Possible reasons for this discrepancy are discussed.     

The proteoliposomal steady state. Effect of size, capacitance and membrane permeability on cytochrome-oxidase-induced ion gradients.

1. The flux pathways for H+ and K+ movements into and out of proteoliposomes incorporating cytochrome c oxidase have been investigated as a function of the electrical and geometrical properties of the vesicles. 2. The respiration-induced pH gradient (delta pH) and membrane potential (delta psi) are mutually dependent and individually sensitive to the permeability properties of the membrane. A lowering or abolition of delta psi by the addition of valinomycin increased the steady-state level of delta pH. Conversely, removal of delta pH by the addition of nigericin resulted in a higher steady-state delta psi. 3. Vesicles prepared by sonication followed by centrifugation maintained similar pH gradients at steady state to those in vesicles prepared by dialysis, although the time taken to reach steady state was longer. Higher pH gradients can be induced in non-centrifuged sonicated preparations. 4. No significant differences were found in H+ and K+ permeability between proteoliposomes prepared by dialysis or by sonication. The permeability coefficient of the vesicle bilayers for H+ was 6.1 x 10(-4) cm.s-1 and that for K+ was 7.5 x 10(-10) cm.s-1. An initial fast change in internal pH was seen on the addition of external acid or alkali, followed by a slower, ionophore-sensitive, change. The initial fast phase can be increased by the lipid-soluble base dibucaine and the weak acid oleate. In the absence of ionophores, increasing concentrations of oleate increased the rate of H+ translocation to a level similar to that seen in the presence of nigericin. Internal alkalinization could also be induced by oleate upon the addition of potassium sulphate. 5. The initial, pre-steady-state and steady-state delta pH and delta psi changes can be simulated using a model in which the enzyme responds to both delta pH and delta psi components of the protonmotive force. At steady state, the electrogenic entry of K+ is countered by electroneutral exit via a K+/H+ exchange. 6. The permeability coefficient, PH, calculated from H+ flux under steady-state turnover conditions, was approx. 100 times higher than the corresponding 'passive' measurements of PH. Under conditions of oxidase turnover, the vesicles appear to be intrinsically more permeable to protons.     

Membrane-associated cytochrome cy of Rhodobacter capsulatus is an electron carrier from the cytochrome bc1 complex to the cytochrome c oxidase during respiration.

We have recently established that the facultative phototrophic bacterium Rhodobacter capsulatus has two different pathways for reduction of the photooxidized reaction center during photosynthesis (F.E. Jenney and F. Daldal, EMBO J. 12:1283-1292, 1993; F.E. Jenney, R.C. Prince, and F. Daldal, Biochemistry 33:2496-2502, 1994). One pathway is via the well-characterized, water-soluble cytochrome c2 (cyt c2), and the other is via a novel membrane-associated c-type cytochrome named cyt cy. In this work, we probed the role of cyt cy in respiratory electron transport by isolating a set of R. capsulatus mutants lacking either cyt c2 or cyt cy, in the presence or in the absence of a functional quinol oxidase-dependent alternate respiratory pathway. The growth and inhibitor sensitivity patterns of these mutants, their respiratory rates in the presence of specific inhibitors, and the oxidation-reduction kinetics of c-type cytochromes monitored under appropriate conditions demonstrated that cyt cy, like cyt c2, connects the bc1 complex and the cyt c oxidase during respiratory electron transport. Whether cyt c2 and cyt cy are the only electron carriers between these two energy-transducing membrane complexes of R. capsulatus is unknown.     

Transport of basic amino acids by membrane vesicles of Lactococcus lactis.

The uptake of the basic amino acids arginine, ornithine, and lysine was studied in membrane vesicles derived from cells of Lactococcus lactis which were fused with liposomes in which beef heart mitochondrial cytochrome c oxidase was incorporated as a proton motive force (PMF)-generating system. In the presence of ascorbate N,N,N'N'-tetramethylphenylenediamine-cytochrome c as the electron donor, these fused membranes accumulated lysine but not ornithine or arginine under aerobic conditions. The mechanism of energy coupling to lysine transport was examined in membrane vesicles of L. lactis subsp. cremoris upon imposition of an artificial electrical potential (delta psi) or pH gradient or both and in fused membranes of these vesicles with cytochrome c oxidase liposomes in which the delta psi and delta pH were manipulated with ionophores. Lysine uptake was shown to be coupled to the PMF and especially to the delta psi, suggesting a proton symport mechanism. The lysine carrier appeared to be specific for L and D isomers of amino acids with a guanidine or NH2 group at the C6 position of the side chain. Uptake of lysine was blocked by p-chloromercuribenzene sulfonic acid but not by maleimides. Counterflow of lysine could not be detected in L. lactis subsp. cremoris, but in the arginine-ornithine antiporter-containing L. lactis subsp. lactis, rapid counterflow occurred. Homologous exchange of lysine and heterologous exchange of arginine and lysine were mediated by this antiporter. PMF-driven lysine transport in these membranes was noncompetitively inhibited by arginine, whereas the uptake of arginine was enhanced by lysine. These observations are compatible with a model in which circulation of lysine via the lysine carrier and the arginine-ornithine antiporter leads to accumulation of arginine.     

Protonmotive stoichiometry of rat liver cytochrome c oxidase: determination by a new rate/pulse method

The stoichoimetry of vectorial H+ ejection coupled to electron flow through the cytochrome c oxidase (EC 1.9.3.1) of rat liver mitochondria was determined by a new rate/pulse method. This is a modification of the oxygen-pulse method. Electron flow through the oxidase is initiated by adding oxygen to suspensions of anaerobic mitochondria at a known and constant rate. Cytochrome c oxidase was examined directly or in combination with cytochrome c reductase (ubiquinol:ferricytochrome c oxidoreductase). In both cases the----H0+/2e- ratio was found to be constant during the time-course of oxygen reduction, and thus independent of delta pH. The stoichiometries observed were consistent with mechanistic stoichiometries of 2 and 6 for cytochrome c oxidase alone and cytochrome c oxidase together with cytochrome c reductase, respectively. The stoichiometry of cytochrome c reductase alone was also examined, by using ferricyanide in place of oxygen. The results obtained were consistent with the accepted mechanistic stoichiometry of 4 for this enzyme.     

Mobile cytochrome c2 and membrane-anchored cytochrome cy are both efficient electron donors to the cbb3- and aa3-type cytochrome c oxidases during respiratory growth of Rhodobacter sphaeroides

We have recently established that the facultative phototrophic bacterium Rhodobacter sphaeroides, like the closely related Rhodobacter capsulatus species, contains both the previously characterized mobile electron carrier cytochrome c2 (cyt c2) and the more recently discovered membrane-anchored cyt cy. However, R. sphaeroides cyt cy, unlike that of R. capsulatus, is unable to function as an efficient electron carrier between the photochemical reaction center and the cyt bc1 complex during photosynthetic growth. Nonetheless, R. sphaeroides cyt cy can act at least in R. capsulatus as an electron carrier between the cyt bc1 complex and the cbb3-type cyt c oxidase (cbb3-Cox) to support respiratory growth. Since R. sphaeroides harbors both a cbb3-Cox and an aa3-type cyt c oxidase (aa3-Cox), we examined whether R. sphaeroides cyt cy can act as an electron carrier to either or both of these respiratory terminal oxidases. R. sphaeroides mutants which lacked either cyt c2 or cyt cy and either the aa3-Cox or the cbb3-Cox were obtained. These double mutants contained linear respiratory electron transport pathways between the cyt bc1 complex and the cyt c oxidases. They were characterized with respect to growth phenotypes, contents of a-, b-, and c-type cytochromes, cyt c oxidase activities, and kinetics of electron transfer mediated by cyt c2 or cyt cy. The findings demonstrated that both cyt c2 and cyt cy are able to carry electrons efficiently from the cyt bc1 complex to either the cbb3-Cox or the aa3-Cox. Thus, no dedicated electron carrier for either of the cyt c oxidases is present in R. sphaeroides. However, under semiaerobic growth conditions, a larger portion of the electron flow out of the cyt bc1 complex appears to be mediated via the cyt c2-to-cbb3-Cox and cyt cy-to-cbb3-Cox subbranches. The presence of multiple electron carriers and cyt c oxidases with different properties that can operate concurrently reveals that the respiratory electron transport pathways of R. sphaeroides are more complex than those of R. capsulatus.     

Mitochondrial complex I impairment and differential carbon monoxide sensitivity of cytochrome c oxidase in wild type and CMS II mutants of Nicotiana sylvestris

Plant mitochondria unlike their animal counterpart have some unique features with highly branched respiratory chain. The present work was undertaken in order to investigate the effect of loss/dysfunction of plant mitochondrial complex I on the relative flux of electrons through alternative oxidase (AOX) and cytochrome oxidase. Loss of a major subunit of mitochondrial complex I in cytoplasmic male sterile II (CMS II) mutant of Nicotiana sylvestris caused respiratory redox perturbations, as evident from the differential CO sensitivity of cytochrome oxidase. The leaf segments of CMS II mutant when exposed to CO under dark aerobic condition were insensitive to the inhibition of cytochrome oxidase, as against the wild type (WT). The differential CO response of WT and CMS II mutants appeared to be due to differences in the redox state of cytochrome a3 (cyt a3), the terminal electron acceptor during in situ respiration. Cyt a3 appeared to be more in its oxidized form in CMS II and hence unable to form cyt a3-CO complex. Pre-treatment of CMS II leaves with 2,4-dinitrophenol, an uncoupler of oxidative phosphorylation increased the CO response. The slight increase in rotenone-insensitive respiration of CMS II could be attributed partly to enhanced flux of electrons through cytochrome pathway to compensate for the loss of phosphorylation site and partly through AOX, which was induced by nitrate.