Kinetic and thermodynamic resolution of cytochrome c1 and cytochrome c2 from Rhodopseudomonas sphaeroides

Abstract

In chromatophores from Rhodopseudomonas sphaeroides, cytochrome c has several characteristics which suggest that there are two distinct populations. Among these are: (i) A displacement of the a-band absorbance maxima of cyt. c l-l.5 nm to the red in chromatophores relative to purified cyt. c2 [1,2]; (ii) Intact spheroplasts and vesicles derived from them retain -50% of the cyt. c found in cells or chromatophores but this cytochrome is unable to react rapidly with the reaction centers ]2,31; (iii) In the presence of UHDBT the cyt. c oxidation kinetics measured at 551-542 nm show two approximately equal phases with halftimes of -5-10 ps and -200 ,us [2]. Direct biochemical methods have shown that in both cells and chromatophores there are two separate c cytochromes, a bound cytochrome cl (h,, 552 nm) and a soluble cyt. c2 (A,, 550 nm) [3,4]. Here, we present evidence on the kinetic, spectral and thermodynamic resolution of the two c-type cytochromes, and show that both are involved in cyclic electron transport, and function in series. A preliminary report of some of this work has appeared in [5]. pared as in [6] and suspended in buffer (50 mM MOPS, 100 mM KCl, pH 7.0) to -0.2 PM reaction center. Time-resolved flash spectra and cytochrome kinetics were obtained as in [2]; the monochromator was a Hilger and Watts D330, and slits were set at 0.5 mm to give spectral bandwidth of 1.3 nm. Cytochrome c2 kinetic changes were measured from the difference between the kinetics at 550 nm and 554 nm, and cyt. cl was measured at 552-548 nm. The flash redox titrations were performed as in [1,7]. Dark redox titrations were performed as in [8,9] and redox resolved spectra were obtained from full spectra redox titrations as in [lo]. The spectrophotometer used a Hilger and Watts D330, D331 double monochromator, with slits set at 1 mm to give a bandwidth of 1.3 nm. UHDBT was a generous gift of B.L. Trumpower and the antimycin was obtained from Sigma.