Kinetics of reduction of Clostridium pasteurianum rubredoxin by laser photoreduced spinach ferredoxin:NADP+ reductase and free flavins. Electron transfer within a protein-protein complex.
The kinetics of reduction of oxidized Clostridium pasteurianum rubredoxin (Rdox) by free flavin semiquinones generated by the laser flash photolysis technique and by spinach ferredoxin:NADP+ reductase (FNR) semiquinone (also produced by flavin semiquinone reduction) have been investigated under anaerobic conditions. 5-Deazariboflavin semiquinone (5-dRf) rapidly reduces oxidized rubredoxin (Rdox) (k = 3.0 X 10(8) M-1 S-1) and oxidized ferredoxin:NADP+ reductase (FNRox) to the semiquinone level (k = 5.5 X 10(8) M-1 S-1). Lumiflavin semiquinone reduces Rdox more slowly (k = 1.3 X 10(7) M-1 S-1) and is not measurably reactive with FNRox. Absorption difference spectroscopy and difference CD indicate that Rdox and FNRox form a 1:1 complex at low ionic strength (10 mM), which is completely dissociated at higher ionic strength (310 mM). Apparent second order rate constants for reduction of Rdox in its free and complexed state by lumiflavin semiquinone are the same. Reduction of Rdox (both free and complexed) by free FNR semiquinone and intracomplex electron transfer were investigated using 5-dRf as the reductant. At I = 10 mM, a first order rate constant of 2.0 X 10(3) S-1 was obtained, which corresponds to the processes involved in intracomplex electron transfer from FNR semiquinone to Rdox. A second order reaction between free FNR semiquinone and complexed Rdox was also observed to occur (k = 5 X 10(7) M-1 S-1). At I = 310 mM, these reactions are not observed and the reaction of FNR semiquinone with free Rdox is second order (k = 4 X 10(6) M-1 S-1).
D002942:Circular Dichroism; D003013:Clostridium / Q000032:analysis*; D004579:Electron Transport; D005287:Ferredoxin-NADP Reductase / Q000378:metabolism* / Q000528:radiation effects; D005288:Ferredoxins / Q000378:metabolism*; D005415:Flavins / Q000378:metabolism*; D007700:Kinetics; D007834:Lasers; D009247:NADH, NADPH Oxidoreductases / Q000378:metabolism*; D010084:Oxidation-Reduction; D010782:Photolysis; D010944:Plants / Q000201:enzymology*; D011809:Quinones / Q000378:metabolism; D012256:Riboflavin / Q000031:analogs & derivatives / Q000378:metabolism; D012416:Rubredoxins / Q000378:metabolism*; D013053:Spectrophotometry
D005288:Ferredoxins; D005415:Flavins; D011809:Quinones; D012416:Rubredoxins; C001922:lumiflavin; C007228:5-deazariboflavin; D005287:Ferredoxin-NADP Reductase; D009247:NADH, NADPH Oxidoreductases; D012256:Riboflavin