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) [Sétif, P. Q. Y., & Bottin, H. (1994) Biochemistry 33, 8495-8504]. The spectra of these three phases (t1/2 < 1 microsecond, = 13-20 and 103-123 microseconds) have been measured between 460 and 600 nm. All of them are fully consistent with electron transfer from (FA,FB)-, the terminal 4Fe-4S acceptors of PSI, to ferredoxin. Though the three spectra deviate significantly from the spectrum that can be calculated independently for this process, their sum closely matches the calculated spectrum. A detailed examination of these deviations indicates that the intermediate (13-20 microseconds) and slow (103-123 microseconds) first-order phases are associated with two distinct ferredoxin-binding sites on PSI. 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 of this process show that it is triggered by collision of free ferredoxin 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 binding are mutually exclusive. The kinetics of ferredoxin reduction were also studied at pH 5.8, in the absence of salts. Under these conditions, the affinity of ferredoxin for PSI is much higher than at pH 8 (dissociation constant approximately 0.05 microM versus 0.6 microM) and the kinetics of ferredoxin reduction are much faster (a major submicrosecond phase and a single first-order microsecond phase with t1/2 approximately 9 microseconds), whereas a third, slower first-order phase is essentially absent. Two similar first-order components are found for the reduction of spinach ferredoxin by PSI from Synechocystis at pH 8, though the apparent dissociation constant for the latter system is larger (approximately 5 microM). Despite the different affinities of spinach and Synechocystis ferredoxins for the cyanobacterial PSI, similar second-order rate constants are found in both cases at pH 8 [(2-6) x 10(8) M-1 s-1].

Laser flash absorption spectroscopy study of ferredoxin reduction by photosystem I in Synechocystis sp. PCC 6803: evidence for submicrosecond and microsecond kinetics.

The kinetics of reduction of soluble ferredoxin by photosystem I (PSI), both purified from the cyanobacterium Synechocystis sp. PCC 6803, were investigated by flash-absorption spectroscopy between 460 and 600 nm. Most experiments were made with isolated monomeric PSI reaction centers prepared with the detergent beta-dodecyl maltoside. Analysis of absorption transients, in parallel at 480 and 580 nm and under several conditions, shows the existence of three different first-order components in the presence of ferredoxin (t1/2 approximately 500 ns, 20 microseconds, and 100 microseconds). A second-order phase of ferredoxin reduction is also present [k = (2-5) x 10(8) s-1 at pH 8 and at moderate ionic strength]. Similar first-order kinetic components were found with membranes from Synechocystis, with dissolved crystals of trimeric PSI reaction centers from Synechococcus, and also when ferredoxin from Synechocystis is replaced by ferredoxin from Chlamydomonas reinhardtii. The three first-order phases exhibit similar, though not identical, spectra which are consistent with electron transfer from the [4Fe-4S] centers of PSI to the [2Fe-2S] center of ferredoxin and are all attributed to reduction of ferredoxin bound to PSI. At pH 8 and at moderate ionic strength, the dissociation constants associated with each of these components are also similar, with a global value varying between 0.2 and 0.8 microM in different cyanobacterial preparations. The presence of three exponential components is discussed assuming homogeneity of the two partners and using the estimated values for the shortest possible distance of approach of soluble ferredoxin from the different iron-sulfur centers of PSI. It is concluded that the 500-ns phase corresponds to electron transfer from either FA- or FB-, the terminal iron-sulfur acceptors of PSI, to ferredoxin and that the immediate electron donor to ferredoxin is reduced within less than 500 ns. The presence of at least two different types of PSI-ferredoxin complex, all competent in electron transfer, is also deduced from the kinetic behavior.