F Malatesta

Structure and function of a molecular machine: cytochrome c oxidase

Cytochrome c is responsible for over 90% of the dioxygen consumption in the living cell and contributes to the build-up of a proton electrochemical gradient derived by the vectorial transfer of electrons between cytochrome c and molecular oxygen. The metal ions found in cytochrome oxidases play a crucial role in these processes and have been extensively studied. In this review we present and discuss some of the relevant spectroscopic and kinetic properties of the prosthetic groups of cytochrome c oxidase.

Hydrogen peroxide is the end product of oxygen reduction by the terminal oxidase in the marine bacterium Pseudomonas nautica 617

Mitochondrial cytochrome‐c oxidase as well as several bacterial oxidases are known to reduce dioxygen to water. For the first time, a heme‐containing oxidase, the terminal enzyme of the aerobic respiratory system in the marine bacterium Pseudomonas nautica 617, is shown to reduce molecular oxygen only to hydrogen peroxide. Whereas the cell content is well protected from H2O2, by catalase, the possible efflux of H2O2, into seawater could play an important role in the environment.

A new method for the determination of the buffer power of artificial phospholipid vesicles by stopped-flow spectroscopy

Abstract Energy-transducing membranes generate protonchemical gradients, and extrused protons have been measured either potentiometrically or spectroscopically by means of a pH indicator. The determination of the stoichiometry of proton pumps ( H + e − ), such as that of cytochrome c oxidase, is affected by the membrane proton permeability and the intrinsic buffer capacity of the system. In order to assess the buffer power of the system (and therefore allow a better estimate of the H + e − ratio for cytochrome c oxidase), a new calibration method which can be employed directly in the stopped-flow apparatus has been introduced. The method makes use of the trypsin-catalysed hydrolysis of N-α- tosyl- l -arginyl -O- methyl ester producing 1 H + /mol at a velocity which can be calibrated (from milliseconds to seconds) changing trypsin concentration. The buffer capacity of phospholipid vesicles in the presence and absence of ionophores has been determined allowing to distinguish between the outer and inner layers of the artificial phospholipid membrane. The determinations acquire significance with reference to the time course of proton permeability of small unilamellar vesicles in the presence of suitably chosen ionophores.

Modulation of Cytochrome c Oxidase Activity by an Electrical Transmembrane Gradient

where n indicates the number of protons that are vectorially translocated across the mitochondria1 inner membrane and subscripts m and c indicate matrix and intermembrane space, respectively. The value of n has been reported to vary between two and eight, which yields an H+ : eratio of 0.5 to z2.’; however, it is generally said that under optimal conditions the H+ : eratio approaches unity. Because cytochrome oxidase reaction (1) contributes to both components of AFH+, that is A+ (the membrane electrical potential) and ApH (the transmembrane pH difference), a quite important issue, yet to be solved, is which component can operate on cytochrome oxidase activity in such a way as to modulate electron transfer and proton-pumping activity. When cytochrome oxidase, reconstituted into artificial phospholipid vesicles, is exposed to a pulse of ferrocytochrome c in the presence of excess dioxygen, biphasic kinetics are observed at a wavelength (550 nm) monitoring the redox state of cytochrome c.4 At this wavelength, the first fast phase (whose amplitude depends on the total amount of substrate added relative to cytochrome oxidase) accounts for the oxidation of four moles of ferrocytochrome c per mole of cytochrome oxidase. The subsequent slower phase is exponential for the rest of the time course, and its first-order rate constant reflects a property of the enzyme that is populated during

Immunoquantitation of Cytochrome c in Cardiac Perfusate

An indirect three step ELISA has been assessed in order to detect the possible release of cytochrome c, a mitochondrial protein, from isolated and perfused guinea-pig heart. The ELISA described in this study is sufficiently sensitive and accurate to measure extracellular cytochrome c.

Molecular control of cytochrome oxidase activity

Abstract Analysis of data obtained by using fast kinetic techniques on cytochrome c oxidase reconstituted into small unilamellar phospholipid vesicles, allowed us to postulate a model based on the presence of two conformational states of the enzyme. In the light of available experimental knowledge we tentatively correlate the membrane potential Um with the relative amount of the two conformers and thus both with the catalytic efficiency and with the proton pumping activity of the reconstituted enzyme. In our conditions the value of the membrane potential reached after 1 turnover (∼ 70 mV) seems to be sufficient to shift the equilibrium towards the less active and slipping state of cytochrome oxidase.

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...

The Metals of Cytochrome C Oxidase and their Role in the Kinetics of Electron Transfer and Proton Pumping

Cytochrome c oxidase (ferrocytochrome c:02 oxidoreductase, EC 1.9.3.1) is a complex oligomeric metalloprotein incorporated in the mitochondrial inner membrane of eukaryotic cells or in the plasma membrane of bacteria [1,2,3]. This enzyme catalyses the transfer of electrons between two substrates, namely ferrocytochrome c and molecular oxygen. Part of the redox energy involved in this reaction is used by the enzyme to transfer protons from the matrix aqueous space to the intermembrane space of mitochondria [4]. Thus, cytochrome c oxidase is a redox-linked proton pump, which couples redox energy to the endoergonic vectorial transport of protons in line with the chemiosmotic theory.

Subunit Interactions in Cytochrome Oxidase: The Role of Subunit III

Cytochrome c oxidase is an oligomeric membrane protein that catalyzes the oxidation of cytochrome c and the reduction of oxygen to water. The enzyme-mediated proton translocation is also known to be linked to its electron transfer activity either in mitochondria or in artificial phospholipid vesicles (1–4). The protein has been purified from several sources with different procedures, resulting in a variable number of co-purifying subunits (7–12) whose specific functions are still, to a large extent, obscure. According to the latest experimental findings in prokaryotes, however, enzyme activity is linked to the presence of at least the three largest subunits (I, II and III), which are coded by mitochondrial DNA in eukaryotes.