LASER FLASH ABSORPTION-SPECTROSCOPY STUDY OF FERREDOXIN REDUCTION BY PHOTOSYSTEM-I - SPECTRAL AND KINETIC EVIDENCE FOR THE EXISTENCE OF SEVERAL PHOTOSYSTEM-I FERREDOXIN COMPLEXES

The existence of three first-order phases has been previously reported for the reduction of soluble ferredoxin by photosystem I (PSI), both from the cyanobacterium Synechocystis sp. PCC 6803 (at pH 8 and in the presence of salts) [Setif, P. Q. Y., & Bottin, H. (1994) Biochemistry 33, 8495-8504]. The spectra of these three phases (t(1/2) < 1 mu s, = 13-20 and 103-123 mu s) have been measured between 460 and 600 nm. Al l of them are fully consistent with electron transfer from (F-A,F-B)(- ), the terminal 4Fe-4S accepters of PSI, to ferredoxin, Though the thr ee spectra deviate significantly from the spectrum that can be calcula ted independently for this process, their sum closely matches the calc ulated spectrum. A detailed examination of these deviations indicates that the intermediate (13-20 mu s) and slow (103-123 mu s) first-order phases are associated with two distinct ferredoxin-binding sites on P SI. Under the same conditions, a fourth phase of negative amplitude is also observed in the 460-600 nm region, It is ascribed to reoxidation of reduced ferredoxin by an unknown species. The kinetic properties o f this process show that it is triggered by collision of free ferredox in with a preformed PSI-ferredoxin complex, Taking this reaction into account, it is shown that the relative proportions of the three first- order phases of ferredoxin reduction do not depend upon the ferredoxin concentration, indicating that the different sites of ferredoxin bind ing are mutually exclusive, The kinetics of ferredoxin reduction were also studied at pH 5.8, in the absence of salts. Under these condition s, the affinity of ferredoxin for PSI is much higher than at pH 8 (dis sociation constant approximate to 0.05 mu M versus 0.6 mu M) and the k inetics of ferredoxin reduction are much faster (a major submicrosecon d phase and a single first-order microsecond phase with t(1/2) approxi mate to 9 mu s), whereas a third, slower first-order phase is essentia lly absent, Two similar first-order components are found for the reduc tion of spinach ferredoxin by PSI from Synechocystis at pH 8, though t he apparent dissociation constant for the latter system is larger (app roximate to 5 mu M). Despite the different affinities of spinach and S ynechocystis ferredoxins for the cyanobacterial PSI, similar second-or der rate constants are found in both cases at pH 8 [(2-6) x 10(8) M(-1 ) s(-1)].