M T Wilson

Nitric oxide and cellular respiration

Abstract. The role of nitric oxide (NO) as a signalling molecule involved in many pathophysiological processes (e.g., smooth muscle relaxation, inflammation, neurotransmission, apoptosis) has been elaborated during the last decade. Since NO has also been found to inhibit cellular respiration, we review here the available information on the interactions of NO with cytochrome c oxidase (COX), the terminal enzyme of the respiratory chain. The effect of NO on cellular respiration is first summarized to present essential evidence for the fact that NO is a potent reversible inhibitor of in vivo O2 consumption. This information is then correlated with available experimental evidence on the reactions of NO with purified COX. Finally, since COX has been proposed to catalyze the degradation of NO into either nitrous oxide (N2O) or nitrite, we consider the putative role of this enzyme in the catabolism of NO in vivo.

Electron transfer and ligand binding in terminal oxidases Impact of recent structural information

A consensus structure for the active site of terminal oxidases has been recently proposed by Hosler et al. [(1993) J. Bioenerg. Biomem. 25, 121–1351]. We exploit the novel structural information to propose a hypothesis for the large difference in the rate of internal electron transfer found when experiments are started either with the reduced or with the oxidized enzyme. This rationale also allows us to discuss the oxidation state of the prevailing oxygen reacting species with reference to the concentration of the two substrates (oxygen and cytochrome c) and to the structural state of the oxidase.

‘Pulsed’ cytochrome oxidase may be produced without the advent of dioxygen

We have reported [l-3] that the product of the reaction of fully reduced cytochrome oxidase with oxygen (either in excess of, or stoichiometric with the functional unit aus) corresponds to a more active form of the enzyme, which we have termed ‘pulsed’ oxidase. The spectral and kinetic properties of ‘pulsed’ oxidase have been characterized [2-61, and particular attention has been paid to the observation that this state of the enzyme catalyzes the oxidation of reduced cytochrome c more rapidly than the ‘resting’ form [WI. The spectral properties of ‘pulsed’oxidase, as determined in stopped-flow experiments [3,4], are similar to those of the classical ‘oxygenated’ compound(s), originally described by Okunuki and coworkers [7] and subsequently investigated in [8-l 11. In addition its kinetic and spectral properties are independent of the nature of the reductant employed to achieve reduction of the enzyme (whether ascorbate and cytochrome c, or dithionite, or NADH and PMS [2-51). Although it was shown [3] that ‘pulsed’ oxidase, similarly to ‘resting’ [12-l 41, contains 4 oxidizing equivalents, it is not yet known whether dioxygen in any of its redox states is still bound to the enzyme or, more importantly, whether it is the only oxidant capable of yielding ‘pulsed’ oxidase. We now report that anaerobic oxidation of reduced cytochrome oxidase with an excess ferricyanide, in the presence or in the absence of cytochrome c, yields a form of the enzyme which is catalytically more active, like ‘pulsed’, and which undergoes a slow transition back to the ‘resting’ state, similar to that reported for ‘oxygenated’ oxidase [7-l 01. Therefore our experiments show that dioxygen is not unique in producing ‘pulsed’ oxidase.

Single cell microspectroscopy reveals that erythrocytes containing hemoglobin S retain a ‘memory’ of previous sickling cycles

Red blood cells from patients homozygotes for hemoglobin S (HbS) have been studied using a computer‐controlled microspectrophotometer, which allows measurements of spectra and dynamics to be undertaken in a single erythrocyte. Complete photodissociation of HbCO results in polymerization of intracellular deoxyhemoglobin S and deformation of the cell. This is associated with a delayed optical change, which, for the same cell, was found to be highly reproducible between repeated cycles of sickling. Comparison of photographic records and absorbance time courses indicates that an erythrocyte, once having undergone a photochemically induced sickling event, always deforms along the same axis during subsequent cycles. This behaviour implies that the cell retains a ‘memory’ of its previous cycle(s) possibly via slow relaxations of the membrane. In addition, rebinding of CO to intracellular hemoglobin was found to be slower if measured after deformation of the cell, with possible important implications for the pathological mechanism of sickling.

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.

Cytochrome Oxidase in Energy Transduction

Cytochrome-c-oxidase, an integral membrane protein of the inner mitrochondrial membrane, is the terminal enzyme of the respiratory chain. This paper summarizes the role of this enzyme in energy conservation, and presents a possible regulatory model based on ΔμH+ linked conformational changes of the macromolecule.