Light induced H + uptake catalysed by photochemical reaction centres from Rhodopseudomonas spheroides R26

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Volume 35, number 2 FEBS LETTERS September 1973 LIGHT INDUCED H + UPTAKE CATALYSED BY PHOTOCHEMICAL REACTION CENTRES FROM RHODOPSEUDOMONAS SPHEROIDES R26 RJ. COGDELL, R.C. PRINCE and A.R. CROFTS Department of Biochemistry, Medical School, University of Bristol, Bristol, BS8 1 TD, England Received20 July 1973 1. Introduction Although it is now generally accepted that light in- duced H ÷ uptake in bacterial chromatophores reflects the activity of an electrogenic pump, the mechanism of this pump remains a subject of considerable contro- versy [1,2]. Studies on the initial kinetics of H + uptake revealed a small initial burst of H ÷ uptake (rapid H ÷ binding) before the slower 'steady state' uptake [3]. The prop- erties of this rapid H ÷ binding have been studied in de- tail following activation with short flashes of light [4-6]. It has been suggested that I-I + uptake reflects the re- duction of a hydrogen carrying redox component dur- ing electron flow [4, 5]. Recently two important lines of evidence have fa- voured this hypothesis: a) Callis et al. [7] have shown that in Chromatium chromatophores the laser induced rapid I-I + binding has very similar kinetics to the reduc- tion of the secondary electron acceptor; b) Flash in- duced rapid H ÷ binding is attenuated in chromato- phores from Rps. spheroides, Rps. capsulata and Rps. viridis as a redox component, which is distinct from the primary electron acceptor, is chemically reduced before the flash [5,8,9]. The variation with pH of the midpoint of attenuation shows that the reduction of this component involves a H + . The isolation of photochemical reaction centres [10] allows more detailed investigation of this phe- nomenon. If the redox component associated with rap- id H + binding is present in the reaction centres then a light induced H + uptake should still be observed, and the relation between the two should be more easily studied. We have chosen to use photochemical reaction cen- tres prepared by the method of Clayton and Wang [10] since they contain a minimal complement ac- cessory components apart from the photochemical re- actants. 2. Materials and methods Ceils ofRps, spheroides R26 were grown in batch culture and chromatophores prepared from them as previously described [ 1 ]. Photochemical reaction cen- tres were isolated from the chromatophores with the zwitterionic detergent LDAO, according to the meth- of of Clayton and Wang [10]. The concentration of reaction centres was determined using a value for EmM 865 of 113 [10]. The concentration of ubiquinone in the reaction centres was determined from a total lipid extract of the reaction centres according to the method of Griffiths et al. [ 11 ]. Light induced H + uptake was monitored with the pH indicator Phenol red, and recorded in a rapidly re- sponding single beam spectrophotometer [5]. The ex- periments were performed in an anaerobic redox cuvette, (Dutton and Jackson, [12] ) which was flushed with O2-free nitrogen. This minimised the pH drifts normally encountered with unbuffered solutions open to the air. The extent of the pH change was 204 North-Holland Publishing Company - Amsterdam