Unbinding of Oxidized Cytochrome c from Photosynthetic Reaction Center of Rhodobacter sphaeroides Is the Bottleneck of Fast Turnover

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 (eta(-)(1)) at 40 and 100 mM ionic strengths and moderate (eta(-)(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 microM. (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.

Kinetics of the quinone binding reaction at the QB site of reaction centers from the purple bacteria Rhodobacter sphaeroides reconstituted in liposomes

“…The results obtained in this paper can be related to the RC photocycle, when the photochemistry takes place in the presence of an exogenous electron donor able to doubly reduce D + . Gerencser et al [49] have measured the steadystate rate of cytochrome c turnover in detergent, demonstrating that at low ionic strength the reaction of cytochrome c 3+ unbinding from the RC is the rate limiting step of the photocycle (1000 s )1 < k off < 2000 s can easily be obtained in our preparation and would give a quinone pool of Q/RC % 3, which is smaller than the average dimension of the quinone pool in chromatophores [50].…”

“…direct micelles, reverse micelles, and proteoliposomes) comes from the influence played by the surroundings on k in , k out and L AB . For instance, in direct lauryl dimethyl amino N-oxide (LDAO) 49 micelles a decay sum of two exponential is observed [18] with a subsaturating quinone concentration [i.e. a fast phase with a decay constant (k F ¼ k AD ) and a slow phase with a decay constant (k S ) given by Eqn (1)], that can be explained only by considering a slow exchange.…”

Transition state and encounter complex for fast association of cytochrome c 2 with bacterial reaction center

“…After a laser flash, the observed second-order association rate constant k 2 for the reaction of the unbound Cyt has been found to be k 2 Ϸ 10 9 s Ϫ1 ⅐M Ϫ1 , close to the diffusion limit (12,13). The first-order electron-transfer rate constant k e Ϸ 10 6 s Ϫ1 (11, 13) measured for RCs having a bound Cyt is much faster than the dissociation-rate constant, k off Ϸ 10 3 s Ϫ1 (15). Because k 2 ϭ k e k on ͞(k off ϩ k e ) (1), this result brings the second-order rate constant into the diffusion-limited regime where the observed second-order rate constant is approximately equal to the association rate constant k on (k 2 Ϸ k on ).…”

Interprotein electron transfer from cytochromec2to photosynthetic reaction center: Tunneling across an aqueous interface

“…For the wild type, the first-order ET rate constant, k e Ϸ 10 6 s Ϫ1 (10,12), measured for RCs having a bound cyt is much faster than the dissociation rate constant, k off Ϸ 10 3 s Ϫ1 (16). Therefore, the second-order ET process is in the diffusion-limited regime, and the observed second-order rate constant is approximately equal to the association rate constant, k on (k 2 Ϸ k on ).…”