THE FLASH-INDUCED TURNOVER OF CYTOCHROME-B-563, CYTOCHROME-F AND PLASTOCYANIN IN CHLOROPLASTS - MODELS AND ESTIMATION OF KINETIC-PARAMETERS

Flash-induced redox changes of cytochrome b-563, cytochrome f and plastocyanin (PC), and the electrochromic response from chloroplast suspensions in reducing conditions (added dichlorophenyldimethylurea, exogenous quinol, anaerobic) were measured in the time range 0-20 ms by deconvoluting absorbance changes at appropriate wavelengths. No response attributable to cytochrome b-559 was observed. Various dimethylbenzoquinols with different substituents on the 6-position of the benzene ring gave similar kinetics for these processes. There was no significant effect on cytochrome b-563, f or PC kinetics of adding nonactin to decay the transthylakoid electric potential difference. These data, together with comparable data for proton deposition, were used in a parameter optimisation procedure, the Inverse Method, to produce rate coefficients for some of the partial reactions occurring when cytochrome f is oxidised by plastocyanin and quinol is subsequently oxidised by cytochrome b/f complexes. Models such as the Q-cycle or semiquinone (SQ) cycle were used to formulate differential equations describing the time-dependencies of various forms of the cytochrome b/f complex containing reduced or oxidised cytochrome f, Rieske centre, cytochrome b-563, and so forth. The Inverse Method minimised the error between data and corresponding model predictions by adjusting parameter values. A model with the two b-563 cytochromes not directly connected electronically was unsatisfactory; Q-cycle and SQ-cycle models could not be differentiated by the available data. A Q-cycle model gave a close match in all respects between data and model predictions using 8 rate coefficients. The following average, reduced rate-coefficients (s-1) were estimated for the chloroplast cytochrome b/f complex under the conditions used: k(FP) (between cytochrome f and PC) 2000; k(PF) (reverse) 220; k(QR) (between quinol and Rieske centre) 200; k(HQ) ((between cytochrome b-563 (high potential) and quinone at n-sites) 380; k(QH) (reverse) 150. The Rieske centre and cytochrome f appeared to be in rapid equilibrium, with an equilibrium constant of about 3, Rieske positive to f.