Control Of Cytochrome Research Articles

Human Energy Expenditure is in Control of Cytochrome C Oxidase Subunit IV‐2

BackgroundThe molecular mechanisms of the pronounced between subject variation in human resting metabolic rate (RMR) remains elusive. The terminal enzyme of the mitochondrial respiratory chain, Cytochrome C oxidase (COX) plays a key role in the control of mitochondrial respiration. There are a number of regulatory mechanisms controlling COX activity and proton pump efficiency. A shift of COX subunit composition may also be a part of the regulation of the enzyme. Recent in vitro studies of COX IV‐2 has implicated this isoform in the cellular response to hypoxia and oxidative stress. The aim of this study was to investigate whether COX IV‐2 is involved in the regulation of respiration in vivo and explains the pronounced individual variations in resting RMR.MethodsIndirect calorimetry was used to measure resting metabolic rate in healthy subjects. Skeletal muscle biopsies were extracted from vastus lateralis for isolation of myogenic satellite cells and protein analysis. Myotubes were transfected with an expression vector in order to achieve over expression of COX IV‐2 and with siRNA for knocking down COX IV‐1 (COX IV‐1−/2+). The control cells were transfected with a control vector and siRNA respectively. Respirometric measurements and hydrogen peroxide production were assessed simultaneously in an oxygraph (O2‐K, Oroboros) equipped with a spectrofluorometer by using the fluorophore Amplex®UltraRed.ResultsHere we show that COX IV‐2 protein is expressed in skeletal muscle harvested from healthy humans with a large inter‐individual difference in the COX IV‐2/COX IV‐1 ratio. A strong inverse correlation was found between the COX IV‐2/COX IV‐1 ratio and RMR measured in the same individuals. The same pattern was observed in the primary human skeletal myotubes overexpressing COX IV‐2 with knocked down COX IV‐1, showing a >60% lower basal cell respiration and a >50% reduction in LEAK respiration. The latter suggests either a lower proton conductance or a reduction in proton slip at COX. In addition, reduced cellular H2O2 production was observed both during LEAK and basal cell respiration.ConclusionThese results suggest an important role of the mitochondrial subunit isoform COX IV‐2 in control of energy expenditure and mitochondrial reactive oxygen species homeostasis in humans.Support or Funding InformationThis study was supported by grants from the Swedish Research Council, Swedish Heart and Lung Foundation, Stockholm City Council (ALF), Swedish National Centre for Research in Sports and funds for the Karolinska Institutet.

Control of cytochrome c redox reactivity through off-pathway modifications in the protein hydrogen-bonding network

Measurements of photoinduced Fe(2+)-to-Ru(3+) electron transfer (ET), supported by theoretical analysis, demonstrate that mutations off the dominant ET pathways can strongly influence the redox reactivity of cytochrome c. The effects arise from the change in the protein dynamics mediated by the intraprotein hydrogen-bonding network.

Role of membrane potential on the control of cytochrome c oxidase over respiration in intact hepatoma HepG2 cells

Role of membrane potential on the control of cytochrome c oxidase over respiration in intact hepatoma HepG2 cells

Fatty acids as modulators of cytochrome c oxidase in proteoliposomes.

The control of cytochrome c oxidase turnover in proteoliposomes by membrane potential (delta psi) and by pH gradient (delta pH) is probably kinetic in nature, and inhibition by valinomycin and stimulation by nigericin indicate that delta pH exerts a greater influence than does an equivalent delta psi. Oleic acid at 100 microM removes all delta psi and delta pH control, whereas a similar concentration of palmitic acid increases turnover but does not completely abolish control. Valinomycin acts synergistically with both fatty acids, indicating that the latter can act as H+/K+ exchangers, but neither fatty acid alone markedly affects delta pH, showing that they cannot fully mimic nigericin. Oleate, but not palmitate, diminishes delta psi, and can move electrophoretically as oleate anion. Submicromolar palmitic acid concentrations partly stimulate turnover in delta psi- and delta pH-controlled proteoliposomes, as reported by Labonia, Muller and Azzi [(1988) Biochem. J. 254, 130-145], which might represent a direct effect on cytochrome c oxidase. The ubiquity of fatty acids in biological membranes suggests that these substances might be responsible for limiting respiratory control and enzyme activity in vivo.

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.

Control of cytochrome c oxidase activity by pH and the electrical potential gradient occurs at separate electron transfer steps and does not require subunit III.

Control of cytochrome c oxidase activity by pH and the electrical potential gradient occurs at separate electron transfer steps and does not require subunit III.

Effect of subunit III removal on control of cytochrome c oxidase activity by pH [Erratum to document cited in CA109(11):88666z

Effect of subunit III removal on control of cytochrome c oxidase activity by pH [Erratum to document cited in CA109(11):88666z

Effect of subunit III removal on control of cytochrome c oxidase activity by pH.

Studies were undertaken to assess the postulated involvement of subunit III in the proton-linked functions of cytochrome c oxidase. The effect of pH on the steady-state kinetic [corrected] parameters of subunit III containing and subunit III depleted cytochrome oxidase was determined by using beef heart and rat liver enzymes reconstituted into phospholipid vesicles. The TNmax and Km values for the III-containing enzyme increase with decreasing pH in a manner quantitatively similar to that reported by Thornstrom et al. [(1984) Chem. Scr. 24, 230-235], giving three apparent pKa values of less than 5.0, 6.2, and 7.8. The maximal activities of the subunit III depleted enzymes (beef heart and rat liver) show a similar dependence on pH, but the Km values are consistently higher than those of the III-containing enzyme, an effect that is accentuated at low pH. The pH dependence of TNmax/Km for both forms of the enzyme (+/- subunit III) indicates that protonation of a group with an apparent pKa of 5.7 lowers the affinity for substrate (cytochrome c) independently of a continued increase in maximal velocity. N,N'-Dicyclohexylcarbodiimide (DCCD) decreases the pH responsiveness of the electron-transfer activity to the same extent in both III-containing and III-depleted enzymes, indicating that this effect is mediated by a peptide other than subunit III. Control of intramolecular electron transfer by a transmembrane pH gradient (or alkaline intravesicular pH) is shown to occur in cytochrome oxidase vesicles with cytochrome c as the electron donor, in agreement with results of Moroney et al. [(1984) Biochemistry 23, 4991-4997] using hexaammineruthenium(II) as the reductant.(ABSTRACT TRUNCATED AT 250 WORDS)

Regulation of the induction of a cytochrome P-450 prostaglandin omega-hydroxylase by pregnancy in rabbit lung.

The mechanism of induction of an adult rabbit cytochrome P-450, prostaglandin (PG) omega-hydroxylase (P-450PG-omega), during pregnancy has been investigated. This P-450 isozyme regiospecifically hydroxylates PGE1, PGA1, and PGF2 alpha at carbon-20 (the omega position). The specific activity of this enzyme is induced from 0.07 nmol of 20-OH-PGE1 to 3.05 nmol of 20-OH-PGE1 formed per min per mg of microsomal protein in the lungs of 25- to 28-day pregnant rabbits as compared to nonpregnant rabbits. Immunoblotting studies with a polyclonal antibody raised against this P-450 have shown that there is a concomitant gestational age-dependent increase in the amount of P-450PG-omega microsomal protein accompanying the increase of omega-hydroxylase activities. Within 3 days postpartum, both omega-hydroxylase activity and the amount of immunodetectable P-450PG-omega drop precipitously to near control levels. In vitro translation of total cellular RNA, extracted from the lungs of pregnant rabbits at various days throughout gestation, and immunoprecipitation of newly synthesized P-450PG-omega demonstrated a similar gestational age-dependent increase in translatable P-450PG-omega mRNA, as was observed with omega-hydroxylase activity and P-450PG-omega protein levels. These data suggest that the induction of this P-450 may occur at the transcriptional level and, furthermore, that control of cytochrome P-450PG-omega expression in the rabbit lung is tightly regulated at both protein and mRNA levels.

The mechanism by which oxygen and cytochrome c increase the rate of electron transfer from cytochrome a to cytochrome a3 of cytochrome c oxidase.

When cytochrome c oxidase is isolated from mitochondria, the purified enzyme requires both cytochrome c and O2 to achieve its maximum rate of internal electron transfer from cytochrome a to cytochrome a3. When reductants other than cytochrome c are used, the rate of internal electron transfer is very slow. In this paper we offer an explanation for the slow reduction of cytochrome a3 when reductants other than cytochrome c are used and for the apparent allosteric effects of cytochrome c and O2. Our model is based on the conventional understanding of cytochrome oxidase mechanism (i.e. electron transfer from cytochrome a/CuA to cytochrome a3/CuB), but assumes a relatively rapid two-electron transfer between cytochrome a/CuA and cytochrome a3/CuB and a thermodynamic equilibrium in the "resting" enzyme (the enzyme as isolated) which favors reduced cytochrome a and oxidized cytochrome a3. Using the kinetic constants that are known for this reaction, we find that the activating effects of O2 and cytochrome c on the rate of electron transfer from cytochrome a to cytochrome a3 conform to the predictions of the model and so provide no evidence of any allosteric effects or control of cytochrome c oxidase by O2 or cytochrome c.

Control of cytochrome P1-450 gene expression by dioxin.

The environmental contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) may produce its effects by altering gene expression in susceptible cells. In mouse hepatoma cells, TCDD induces the transcription of the cytochrome P1-450 gene, whose product, aryl hydrocarbon hydroxylase, contributes both to the detoxification and to the metabolic activation of carcinogenic polycyclic aromatic hydrocarbons. A DNA fragment containing sequences flanking the 5' end of the cytochrome P1-450 gene was isolated and analyzed. This DNA fragment contains a cis-acting control element with at least three functional domains: a putative promoter, an inhibitory domain upstream from the promoter that blocks its function, and a TCDD-responsive domain still farther (1265 to 1535 base pairs) upstream of the promoter. These findings, together with results from earlier studies, imply that transcription of the cytochrome P1-450 gene is under both positive and negative control by at least two trans-acting regulatory factors.

AN INVERSE RELATIONSHIP OF THE EFFECTS OF OXYGEN AND IRON ON THE PRODUCTION OF FLUORESCEIN AND CYTOCHROME C BY PSEUDOMONAS FLUORESCENS.

IT has been suggested by Burton et al. that fluorescin, a yellow-green fluorescent bacterial pigment, is related to respiratory enzymes. Observations from this laboratory on the control of cytochrome c production in Pseudomonas fluorescens2–4 are in general accord with Burton's hypothesis, and suggest that fluorescin is an alternative product of porphyrin metabolism.