Emilio D'Itri

On the Mechanism of Inhibition of Cytochrome c Oxidase by Nitric Oxide

The mechanism of inhibition of cytochrome (cyt) c oxidase by nitric oxide (NO) has been investigated by stopped flow transient spectroscopy and singular value decomposition analysis. Following the time course of cyt c oxidation at different O2/NO ratios, we observed that the onset of inhibition: (i) is fast and at a high NO concentration is complete during the first turnover; (ii) is sensitive to the O2/NO ratio; and (iii) is independent of incubation time of the oxidized enzyme with NO. Analysis of the reaction kinetics and computer simulations support the conclusion that inhibition occurs via binding of NO to a turnover intermediate with a partially reduced cyt a3-CuB binuclear center. The inhibited enzyme has the optical spectrum typical of NO bound to reduced cyt a3. Reversal of inhibition in the presence of O2 does not involve a direct reaction of O2 with NO while bound at the binuclear center, since recovery of activity occurs at the rate of NO dissociation (k = 0.13 s−1), as determined in the absence of O2 using hemoglobin as a NO scavenger. We propose that removal of NO from the medium is associated with reactivation of the enzyme via a relatively fast thermal dissociation of NO from the reduced cyt a3-CuB center.

Internal Electron Transfer in Cu-Heme Oxidases THERMODYNAMIC OR KINETIC CONTROL?

We present novel experimental evidence that, starting with the oxidized enzyme, the internal electron transfer in cytochrome c oxidase is kinetically controlled. The anaerobic reduction of the oxidized enzyme by ruthenium hexamine has been followed in the absence and presence of CO or NO, used as trapping ligands for reduced cytochrome a 3. In the presence of NO, the rate of formation of the cytochromea 3 2+-NO adduct is independent of the concentration of ruthenium hexamine and of NO, indicating that in the oxidized enzyme cytochrome a anda 3 are not in very rapid redox equilibrium; on the other hand, CO proved to be a poor “trapping” ligand. We conclude that the intrinsic rate constant for a →a 3 electron transfer in the oxidized enzyme is 25 s−1. These data are discussed with reference to a model (Verkhovsky, M. I., Morgan, J. E., and Wikström, M. (1995)Biochemistry 34, 7483–7491) in which H+diffusion and/or binding at the binuclear site is the rate-limiting step in the reduction of cytochrome a 3 in the oxidized enzyme.

Spectral analysis of cytochromes in rat heart myocytes: Transient and steady-state photodiode array spectrophotometry measurements

Myocytes prepared from rat heart have been studied by optical spectroscopy using a photodiode array spectrophotometer adapted to a stopped flow apparatus (PASF). The isolated cells were viable for 3-4 h (i.e., over the total time of the experiments), as tested employing morphological parameters of cell damage, reactivity toward trypan blue, and the ability to use succinate in the absence and presence of digitonin. Respiration was activated by addition of sodium ascorbate and tetramethyl-para-phenylenediamine (TMPD) as exogenous reductants, in order to single out the contributions of cytochrome c and cytochrome c oxidase among the complexes of the mitochondrial respiratory chain. TMPD was shown to be freely permeable across cytoplasmic and mitochondrial membranes, with a measured KD = 0.9 mM. The use of singular value decomposition analysis coupled to PASF acquisition proved very powerful in resolving statically and kinetically, in the millisecond time region, the spectral contributions of the cytochromes. Spectral analysis was improved by adding carbon monoxide at concentrations which did not affect cytochrome c oxidase activity, but kept myoglobin fully saturated (and thus uninfluential to absorbance changes).

Reconstitution of cytochrome c oxidase into phospholipid vesicles: Effect of detergents

Abstract Cytochrome c oxidase vesicles prepared using enzyme preparations subjected to cycles of freezing and thawing (+20 to −20°C) before reconstitution, display a decrease in respiratory control ratio (RCR); if the protein is incubated with detergents before reconstitution, a higher RCR value is restored. This effect is attributed to a detergent-mediated optimization of the structural assembly of the proteo-membrane unit occurring at the early stages of reconstitution. The same type of experiment carried out at different temperatures showed that incubation at 35°C for 30 min leads to a severe, irreversible loss of RCR.

Liposomal and Mitochondrial Cytochrome Oxidase Display Similar Bioenergetic Properties

AbstractCytochrome c oxidase, the terminal electron acceptor of the respiratory chain of mitochondria, is an integral membrane protein. The bioenergetic properties of cytochrome oxidase can be studied only when the macromolecule is inserted in a phospholipid bilayer, either in situ or after reconstitution into liposomal membranes. Reintegration of purified cytochrome oxidase in liposomes allows quantitative tests of mechanistic hypothesis concerning the functional properties of the enzyme. Small unilamellar vesicles are prepared by sonication of purified soybean asolectin, and reconstitution of cytochrome oxidase in the bilayer is carried out according to the cholate/dialysis procedure. The proteoliposomes are shown to mimick the mitochondrial state of the enzyme in so far as liposomal cytochrome oxidase : a) displays the same vectorial orientation, the cytochrome c binding site being externally exposed, b) pumps protons in the physiological inside/outside direction, and c) is functionally controlled by t...