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ABSTRACT: Aerobic respiration in bacteria, Archaea, and mitochondria is performed by oxygen reductase members of the heme-copper oxidoreductase superfamily. These enzymes are redox-driven proton pumps which conserve part of the free energy released from oxygen reduction to generate a proton motive force. The oxygen reductases can be divided into three main families based on evolutionary and structural analyses (A-, B- and C-families), with the B- and C-families evolving after the A-family. The A-family utilizes two proton input channels to transfer protons for pumping and chemistry, whereas the B- and C-families require only one. Generally, the B- and C-families also have higher apparent oxygen affinities than the A-family. Here we use whole cell proton pumping measurements to demonstrate differential proton pumping efficiencies between representatives of the A-, B-, and C-oxygen reductase families. The A-family has a coupling stoichiometry of 1 H(+)/e(-), whereas the B- and C-families have coupling stoichiometries of 0.5 H(+)/e(-). The differential proton pumping stoichiometries, along with differences in the structures of the proton-conducting channels, place critical constraints on models of the mechanism of proton pumping. Most significantly, it is proposed that the adaptation of aerobic respiration to low oxygen environments resulted in a concomitant reduction in energy conservation efficiency, with important physiological and ecological consequences.
Proceedings of the National Academy of Sciences 08/2011; 108(34):14109-14. · 9.68 Impact Factor
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ABSTRACT: The respiratory chain of Vibrio cholerae contains three bd-type quinol oxygen reductases as well as one cbb(3) oxygen reductase. The cbb(3) oxygen reductase has been previously isolated and characterized; however, the natural mobile electron donor(s) that shuttles electrons between the bc(1) complex and the cbb(3) oxygen reductase is not known. The most likely candidates are the diheme cytochrome c(4) and monoheme cytochrome c(5), which have been previously shown to be present in the periplasm of aerobically grown cultures of V. cholerae. Both cytochromes c(4) and c(5) from V. cholerae have been cloned and expressed heterologously in Escherichia coli. It is shown that reduced cytochrome c(4) is a substrate for the purified cbb(3) oxygen reductase and can support steady state oxygen reductase activity of at least 300 e(-1)/s. In contrast, reduced cytochrome c(5) is not a good substrate for the cbb(3) oxygen reductase. Surprisingly, the dependence of the oxygen reductase activity on the concentration of cytochrome c(4) does not exhibit saturation. Global spectroscopic analysis of the time course of the oxidation of cytochrome c(4) indicates that the apparent lack of saturation is due to the strong dependence of K(M) and V(max) on the concentration of oxidized cytochrome c(4). Whether this is an artifact of the in vitro assay or has physiological significance remains unknown. Cyclic voltammetry was used to determine that the midpoint potentials of the two hemes in cytochrome c(4) are 240 and 340 mV (vs standard hydrogen electrode), similar to the electrochemical properties of other c(4)-type cytochromes. Genomic analysis shows a strong correlation between the presence of a c(4)-type cytochrome and a cbb(3) oxygen reductase within the beta- and gamma-proteobacterial clades, suggesting that cytochrome c(4) is the likely natural electron donor to the cbb(3) oxygen reductases within these organisms. These would include the beta-proteobacteria Neisseria meningitidis and Neisseria gonnorhoeae, in which the cbb(3) oxygen reductases are the only terminal oxidases in their respiratory chains, and the gamma-proteobacterium Pseudomonas stutzeri.
Biochemistry 09/2010; 49(35):7494-503. · 3.42 Impact Factor
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ABSTRACT: Circadian clocks comprise a cyclic series of dynamic cellular states, characterized by the changing availability of substrates that alter clock time when activated. To determine whether circadian clocks, like the cell cycle, exhibit regulation by key phosphorylation events, we examined endogenous kinase regulation of timekeeping in the mammalian suprachiasmatic nucleus (SCN). Short-term inhibition of PKG-II but not PKG-Ibeta using antisense oligodeoxynucleotides delayed rhythms of electrical activity and Bmal1 mRNA. Phase resetting was rapid and dynamic; inhibition of PKG-II forced repetition of the last 3.5 hr of the cycle. Chronic inhibition of PKG-II disrupted electrical activity rhythms and tonically increased Bmal1 mRNA. PKG-II-like immunoreactivity was detected after coimmunoprecipitation with CLOCK, and CLOCK was phosphorylated in the presence of active PKG-II. PKG-II activation may define a critical control point for temporal progression into the daytime domain by acting on the positive arm of the transcriptional/translational feedback loop.
Neuron 09/2004; 43(4):539-49. · 14.74 Impact Factor
