The proton-pumping site of cytochrome c oxidase: a model of its structure and mechanism

Cytochrome c oxidase is an electron-transfer driven proton pump. In this paper, we propose a complete chemical mechanism for the enzyme's proton-pumping site. The mechanism achieves pumping with chemical reaction steps localized at a redox center within the enzyme; no indirect coupling through protein conformational changes is required. The proposed mechanism is based on a novel redox-linked transition metal ligand substitution reaction. The use of this reaction leads in a straightforward manner to explicit mechanisms for achieving all of the processes previously determined (Blair, D.F., Gelles, J. and Chan, S.I. (1986) Biophys. J. 50, 713-733) to be needed to accomplish redox-linked proton pumping. These processes include: (1) modulation of the energetics of protonation/deprotonation reactions and modulation of the energetics of redox reactions by the structural state of the pumping site; (2) control of the rates of the pump's redox reactions with its electron-transfer partners during the turnover cycle (gating of electrons); and (3) regulation of the rates of the protonation/deprotonation reactions between the pumping site and the aqueous phases on the two sides of the membrane during the reaction cycle (gating of protons). The model is the first proposed for the cytochrome oxidase proton pump which is mechanistically complete and sufficiently specific that a realistic assessment can be made of how well the model pump would function as a redox-linked free-energy transducer. This assessment is accomplished via analyses of the thermodynamic properties and steady-state kinetics expected of the model. These analyses demonstrate that the model would function as an efficient pump and that its behavior would be very similar to that observed of cytochrome oxidase both in the mitochondrion and in purified preparations. The analysis presented here leads to the following important general conclusions regarding the mechanistic features of the oxidase proton pump. (1) A workable proton-pump mechanism does not require large protein conformational changes. (2) A redox-linked proton pump need not display a pH-dependent midpoint potential, as has frequently been assumed. (3) Mechanisms for redox-linked proton pumps that involve transition metal ligand exchange reactions are quite attractive because such reactions readily lend themselves to the linked gating processes necessary for proton pumping.     

Redox-linked proton translocation in cytochrome oxidase: the importance of gating electron flow. The effects of slip in a model transducer.

In at least one component of the mitochondrial respiratory chain, cytochrome c oxidase, exothermic electron transfer reactions are used to drive vectorial proton transport against an electrochemical hydrogen ion gradient across the mitochondrial inner membrane. The role of the gating of electrons (the regulation of the rates of electron transfer into and out of the proton transport site) in this coupling between electron transfer and proton pumping has been explored. The approach involves the solution of the steady-state rate equations pertinent to proton pump models which include, to various degrees, the uncoupled (i.e., not linked to proton pumping) electron transfer processes which are likely to occur in any real electron transfer-driven proton pump. This analysis furnishes a quantitative framework for examining the effects of variations in proton binding site pKas and metal center reduction potentials, the relationship between energy conservation efficiency and turnover rate, the conditions for maximum power output or minimum heat production, and required efficiency of the gating of electrons. Some novel conclusions emerge from the analysis, including: An efficient electron transfer-driven proton pump need not exhibit a pH-dependent reduction potential; Very efficient gating of electrons is required for efficient electron transfer driven proton pumping, especially when a reasonable correlation of electron transfer rate and electron transfer exoergonicity is assumed; and A consideration of the importance and possible mechanisms of the gating of electrons suggests that efficient proton pumping by CuA in cytochrome oxidase could, in principle, take place with structural changes confined to the immediate vicinity of the copper ion, while proton pumping by Fea would probably require conformational coupling between the iron and more remote structures in the enzyme. The conclusions are discussed with reference to proton pumping by cytochrome c oxidase, and some possible implications for oxidative phosphorylation are noted.     

Temperature dependence of the reduction potential of CuA in carbon monoxide inhibited cytochrome c oxidase

The temperature dependence of the reduction potential of the CuA site in carbon monoxide inhibited cytochrome c oxidase has been measured with a spectroelectrochemical method adapted to the relatively weak near-infrared absorption of this copper ion. These measurements, together with parallel measurements on the 604-nm absorption due to Fea, indicate that an interaction between CuA and Fea causes the reduction potential for one of these sites to be decreased by approximately 40 mV upon reduction of the other. The temperature dependence of the CuA reduction potential indicates a relatively large and negative standard entropy of reduction of CuA (delta So' = -48.7 +/- 2.3 eu). Possible implications of the intersite redox interaction and the large standard entropy of reduction of the CuA site are discussed.     

Effect of habitable space on substrate selection by Paragnetina media (Walker) (Plecoptera: Perlidae)

A biological measure of space available within substrates was used as an index to examine substrate selection by the stonefly nymph Paragnetina media (Walker). Physical measures, such as total surface area of substrate, have not worked well in the past in explaining distribution of aquatic invertebrates. Although analysis of habitable space within substrate did not explain selection completely, the technique provided a precise measure and might be a more rigorous means by which substrate selection could be examined.     

Chemical and spectroscopic evidence for the formation of a ferryl Fea3 intermediate during turnover of cytochrome c oxidase

When partially reduced cytochrome c oxidase samples are reoxidized with dioxygen, an EPR-silent dioxygen intermediate, which is at the three-electron level of dioxygen reduction, is trapped at the dioxygen reduction site. The intermediate has novel spectral features at 580 and 537 nm. Combined optical and EPR results reveal that this intermediate reacts rapidly with CO at 277-298 K causing the abolition of the 580/537 mm features and the appearance of a rhombic CuB EPR signal. A ferryl Fea3, or an intermediate at the same formal level of oxidation, is proposed to oxidize CO to CO2 producing an EPR-detectable CuB adjacent to a low-spin ferrous Fea3-dioxygen (or carbon monoxide) adduct.     

Carbon isotopic fractionation in heterotrophic microbial metabolism.

Differences in the natural-abundance carbon stable isotopic compositions between products from aerobic cultures of Escherichia coli K-12 were measured. Respired CO2 was 3.4% depleted in 13C relative to the glucose used as the carbon source, whereas the acetate was 12.3% enriched in 13C. The acetate 13C enrichment was solely in the carboxyl group. Even though the total cellular carbon was only 0.6% depleted in 13C, intracellular components exhibited a significant isotopic heterogeneity. The protein and lipid fractions were -1.1 and -2.7%, respectively. Aspartic and glutamic acids were -1.6 and +2.7%, respectively, yet citrate was isotopically identical to the glucose. Probable sites of carbon isotopic fractionation include the enzyme, phosphotransacetylase, and the Krebs cycle.     

A three-dimensional computational simulation of some sources of measurement artifact in microvascular pulsed ultrasound Doppler velocimetry

Recent applications of 20 MHz pulsed ultrasound Doppler velocimetry (PUDVM) in microsurgical research have necessarily employed piezoelectric crystals whose diameter is not negligible compared to the lumen size (1-2 mm) of many vessels of interest. A three-dimensional numerical model was developed to explore relationships between actual and detected flow field parameters, for (steady) Poiseuille flow, when appreciable velocity gradients exist within the PUDVM sample volume. Validation studies showed that highly accurate velocity profiles could be obtained in the limiting case of a very small sample volume (0.1 mm radius), but that for currently employed crystals (approximately equal to 0.5 mm radius) there was appreciable underestimation of the centersteam velocity, and appreciable overestimation of the flow stream diameter. Errors in perceived velocity and flow rate were found to be relatively insensitive to perturbations in the sample volume thickness, in the size of the sampling range increment, or in the angle of insonation beam divergence. By contrast, these apparent flow parameters were found to be very sensitive to perturbations of sample volume diameter or of the Doppler angle. Small variations in the degree of partial sample volume overlap of the flowstream periphery were shown to be capable of causing large fluctuations in apparent flow stream diameter.     

Deurithitrema gingae n.g., n.sp. (Digenea: Plagiorchioidea) from the kidneys of a salt-water crocodile (Crocodylus porosus Schneider) from Australia

Summary The name Deurithitrema gingae n. g., n. sp. is proposed for worms from the kidneys of one Crocodylus porosus from Darwin, Northern Territory, Australia. The following combination of characters distinguishes the new genus from all others: prepharynx very short; oesophagus absent; caeca reaching to posterior end of body; testes tandem in middle third of body, contiguous or amost so; cirrus-sac large, over-reaches ventral sucker, contains bipartite seminal vesicle, prostatic regions and ejaculatory duct with spines around its opening into genital atrium; genital pore immediately in front of ventral sucker; uterus coiled to posterior end of body; vitellaria lateral from ventral sucker to slightly behind testes; excretory vesicle small, receiving a single ventro-lateral duct from each side behind end of uterine loops. Deurithitrema belongs to the superfamily Plagiorchioidea Dollfus, 1930. It superficially resembles a number of genera, but its relationships within the superfamily are obscure.