Coupling of proton flow to ATP synthesis in Rhodobacter capsulatus: F0F1-ATP synthase is absent from about half of chromatophores

Abstract

F0F1-ATP synthase (H+-ATP synthase, F0F1) utilizes the transmembrane protonmotive force to catalyze the formation of ATP from ADP and inorganic phosphate (P-i). Structurally the enzyme consists of a membrane-embedded proton-translocating F-0 portion and a protruding hydrophilic F-1 part that catalyzes the synthesis of ATP. In photosynthetic purple bacteria a single turnover of the photosynthetic reaction centers (driven by a short saturating flash of light) generates protonmotive force that is sufficiently large to drive ATP synthesis. Using isolated chromatophore vesicles of Rhodobacter capsulatus, we monitored the flash induced ATP synthesis (by chemoluminescence of luciferin/luciferase) in parallel to the transmembrane charge transfer through F0F1 (by following the decay of electrochromic bandshifts of intrinsic carotenoids). With the help of specific inhibitors of F-1 (efrapeptin) and of F-0 (venturicidin), we decomposed the kinetics of the total proton flow through F0F1 into (i) those coupled to the ATP synthesis and (ii) the de-coupled proton escape through F-0. Taking the coupled proton flow, we calculated the H+/ATP ratio; it was found to be 3.3 /- 0.6 at a large driving force (after one saturating flash of light) but to increase up to 5.1 /- 0.9 at a smaller driving force (after a half-saturating flash). From the results obtained, we conclude that our routine chromatophore preparations contained three subsets of chromatophore vesicles. Chromatophores with coupled F0F1 dominated in fresh material. Freezing/thawing or pre-illumination in the absence of ADP and P-1 led to an increase in the fraction of chromatophores with at least one de-coupled F-0(F-1). The disclosed fraction of chromatophores that lacked proton-conducting F-0(F-1) (approx. 40% of the total amount) remained constant upon these treatments. (C) 2001 Elsevier Science B.V. All rights reserved.