The major pathways for energy conversion in living cells are respiration and photosynthesis. The underlying mechanisms have many common features. The coupling between electron transfer and phosphorylation is through a proton circuit, and several of the enzymes, in particular the H+-ATP synthase, and the quinol oxidizing complexes, show so strong a similarity as to suggest a common evolutionary ancestry. This homology is most striking when mitochondria are compared to photosynthetic bacteria. In the present proposal we will continue and extend our work on mechanisms of energy conservation in the photosynthetic bacteria, taking advantage of the metabolic versatility of the bacteria, the potential for genetic manipulation and protein engineering, and the many experimental advantages arising from the ability to initiate electron transfer by light. The overall long term aim of the project is to understand the mechanism of energy conversion. With the emerging consensus that the mechanism is chemiosmotic. the immediate aims will be to study the molecular mechanisms of individual proton pumps, to investigate the mechanisms by which the proton gradient controls electron transport, and to study the integration of photochemical devices in the proton circuits. Studies of electron transfer in the UQH2:cyt c2 oxidoreductase of Rhodobacter sphaeroides have suggested a detailed mechanism. Spectroscopic techniques will be used to probe the local molecular environment of prosthetic groups in the complex, and protein engineering to analyse the contribution of particular amino acid residues to catalytic mechanism, and the liganding of prosthetic groups. The relation of electron transfer to the proton gradient will be studied by using the electrochromic response of the bulk pigments to follow electrogenic processes. We will study the thermodynamic poise of the redox components and the membrane potential, and the feedback control exerted by the proton gradient. Studies of the flux through the electron transfer chain, and the current across the membrane will provide information about the degree of coupling, and will help to resolve a long standing controversy about local v. delocalized chemiosmotic mechanisms. Other proton pumps and photochemical devices in the bacterial system, and the integration of these into the physiological proton circuit, will be studied.
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