Unbinding of Oxidized Cytochrome c from Photosynthetic Reaction Center of Rhodobacter sphaeroides Is the Bottleneck of Fast Turnover † La ´szlo ´ Gerencse ´r, Ga ´bor Laczko ´, and Pe ´ter Maro ´ti* Department of Biophysics, UniVersity of Szeged, Egyetem utca 2, Szeged, Hungary H-6722 ReceiVed July 7, 1999; ReVised Manuscript ReceiVed September 28, 1999 ABSTRACT: To understand the details of rate limitation of turnover of the photosynthetic reaction center, photooxidation of horse heart cytochrome c by reaction center from Rhodobacter spheroides in detergent dispersion has been examined by intense continuous illumination under a wide variety of conditions of cytochrome concentration, ionic strength, viscosity, temperature, light intensity, and pH. The observed steady-state turnover rate of the cytochrome was not light intensity limited. In accordance with recent findings [Larson, J. W., Wells, T. A., and Wraight, C. A. (1998) Biophys. J. 74 (2), A76], the turnover rate increased with increasing bulk ionic strength in the range of 0-40 mM NaCl from 1000 up to 2300 s -1 and then decreased at high ionic strength under conditions of excess cytochrome and ubiquinone and a photochemical rate constant of 4500 s -1 . Furthermore, we found the following: (i) The contribution of donor (cytochrome c) and acceptor (ubiquinone) sides as well as the binding of reduced and the release of oxidized cytochrome c could be separated in the observed kinetics. At neutral and acidic pH (when the proton transfer is not rate limiting) and at low or moderate ionic strength, the turnover rate of the reaction center was limited primarily by the low release rate of the photooxidized cytochrome c (product inhibition). At high ionic strength, however, the binding rate of the reduced cytochrome c decreased dramatically and became the bottleneck. The observed activation energy of the steady-state turnover rate reflected the changes in limiting mechanisms: 1.5 kcal/mol at 4 mM and 5.7 kcal/mol at 100 mM ionic strength. A similar distinction was observed in the viscosity dependence of the turnover rate: the decrease was steep (η -1 ) at 40 and 100 mM ionic strengths and moderate (η -0.2 ) under low-salt (4 mM) conditions. (ii) The rate of quinone exchange at the acceptor side with excess ubiquinone-30 or ubiquinone-50 was higher than the cytochrome exchange at the donor side and did not limit the observed rate of cytochrome turnover. (iii) Multivalent cations exerted effects not only through ionic strength (screening) but also by direct interaction with surface charge groups (ion-pair production). Heavy metal ion Cd 2+ bound to the RC with apparent dissociation constant of 14 μM. (iv) A two-state model of collisional interaction between reaction center and cytochrome c together with simple electrostatic considerations in the calculation of rate constants was generally sufficient to describe the kinetics of photooxidation of dimer and cytochrome c. (v) The pH dependence of cytochrome turnover rate indicated that the steady-state turnover rate of the cytochrome under high light conditions was not determined by the isoelectric point of the reaction center (pI ) 6.1) but by the carboxyl residues near the docking site. Water-soluble cytochrome c interacts with several redox- active proteins of aerobic and anaerobic respiration (e.g., cytochrome c peroxidase, flavocytochrome b 2 , NO 2 - reduc- tase, N 2 O reductase, NO reductase, cellobiose oxidase, etc.) and bacterial photosynthesis. The physiological role of cytochrome c 2 in photosynthetic bacteria is well estab- lished: it shuttles electrons between the integral membrane proteins of photochemical reaction center (RC) 1 and the ubiquinol-cyt c 2 oxidoreductase (for reviews, see refs 1-3). These small (12-14 kDa) single-heme proteins are located in the aqueous periplasmic phase outside the intracytoplasmic membrane and inside the outer membrane (cell wall) of the bacterium. As mobile electron carriers, they diffuse between the electron-donating and -accepting reaction sites of these membrane redox proteins along the membrane-aqueous interface and through the aqueous solution. Cytochrome c 2 exports oxidizing equivalents from light- activated RC of purple bacterium Rhodobacter (Rb.) sphaeroi- des. A routine route to study this action is to expose the RC dissolved in detergent or reconstituted in phospholipid vesicles to a brief and saturating flash of light that turns over the RC just once. The RC-cyt c system displays kinetically distinct phases. The fast phase [t 1/2 ∼ 1 μs(4-7)] arises from “proximal” (bound) cytochrome at the time of the flash, † We are grateful for the financial support of the Hungarian Science Foundation (OTKA 17362/95, T30337, and M27903), Foundation of Hungarian Ministry of Education (FKFP 1288 and B-23/1997, FEFA III/1034 and IV/1605, and AMFK 043/98), and NATO (LST.CLG 975754). * Corresponding author. Fax: 36-62-454-121. E-mail: pmaroti@ physx.u-szeged.hu. 1 Abbreviations: cyt 3+ and cyt 2+ , oxidized and reduced cytochrome c, respectively; KD, dissociation constant; LDAO, N,N′-dimethyl dodecylamine N-oxide; 1,2-NQ, 1,2-naphthoquinone; P, bacteriochlo- rophyll dimer that is the primary electron donor in the reaction center protein; QA and QB, primary and secondary quinone, respectively; Rb., Rhodobacter; RC, reaction center; Triton X-100, polyoxyethylene(10) isooctylphenyl ether; UQ6, ubiquinone-30; UQ10, ubiquinone-50. 16866 Biochemistry 1999, 38, 16866-16875 10.1021/bi991563u CCC: $18.00 © 1999 American Chemical Society Published on Web 11/25/1999