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].
Sétif
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PQ
Département de Biologie Cellulaire et Moléculaire, CEA, Gif sur Yvette, France.
Bottin
H
H
D001665:Binding Sites; D000458:Cyanobacteria / Q000378:metabolism; D005288:Ferredoxins / Q000737:chemistry / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D007834:Lasers; D010084:Oxidation-Reduction; D045322:Photosynthetic Reaction Center Complex Proteins / Q000737:chemistry / Q000378:metabolism*; D045331:Photosystem I Protein Complex; D013053:Spectrophotometry; D018724:Spinacia oleracea / Q000378:metabolism
D005288:Ferredoxins; D045322:Photosynthetic Reaction Center Complex Proteins; D045331:Photosystem I Protein Complex