A new system for direct determination of H+/0 stoichiometric ratios during the rapid initiation of a respiratory pulse (developed in the laboratory) was used for extensive studies with two systems. Succinate as electron donor with intact mitochondria activated energy transduction sites II and III. Reduced cytochrome c and mitoplasts isolated site III. In both cases a new phenomenon was observed - a rapid burst in H+/0 ratio peaking within 50 ms and dissipating by 300 ms. When data from 0 to 0.8 s was ignored and the remaining data extrapolated back to zero time on the assumption of lst order kinetics, we confirmed Lehninger's zero time value of 8 for the succinate system as opposed to 6 favored by Wikstrom or 4 favored by Mitchell. However, our further analysis of the extrapolation procedure leads us to conclude that no value obtained by this technique is dependable. We are considering an alternative model for linkage between respiration and proton translocation other than the direct movement of one proton per electron as postulated in the Mitchell hypothesis. In this model there is no fixed ratio for H+/0, but rather the number would be a variable determined solely by the amount of energy liberated by respiration and the number of protons that could be moved against the LambdaMuH+ existing at the instant. Work has continued on the completion and testing of an alternative experimental system for determing early H+/0. The new system does not employ relaxation time corrections for electrodes nor the presence of a carbon monoxide atmosphere.

National Institute of Health (NIH)
National Heart, Lung, and Blood Institute (NHLBI)
Intramural Research (Z01)
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U.S. National Heart Lung and Blood Inst
United States
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Hendler, R W; Drachev, L A; Bose, S et al. (2000) On the kinetics of voltage formation in purple membranes of Halobacterium salinarium. Eur J Biochem 267:5879-90
Joshi, M K; Bose, S; Hendler, R W (1999) Regulation of the bacteriorhodopsin photocycle and proton pumping in whole cells of Halobacterium salinarium. Biochemistry 38:8786-93