激光光还原菠菜铁氧化还原蛋白：NADP⁺还原酶和游离黄素对巴氏梭菌红素还原蛋白的还原动力学。蛋白质-蛋白质复合物内的电子转移。

Kinetics of reduction of Clostridium pasteurianum rubredoxin by laser photoreduced spinach ferredoxin:NADP+ reductase and free flavins. Electron transfer within a protein-protein complex.

作者信息

Przysiecki C T, Bhattacharyya A K, Tollin G, Cusanovich M A

出版信息

J Biol Chem. 1985 Feb 10;260(3):1452-8.

Abstract

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).

摘要

在厌氧条件下，研究了通过激光闪光光解技术产生的游离黄素半醌以及菠菜铁氧化还原蛋白：NADP⁺还原酶（FNR）半醌（也由黄素半醌还原产生）对氧化型巴氏梭菌红素氧还蛋白（Rdox）的还原动力学。5-脱氮核黄素半醌（5-dRf）能迅速将氧化型红素氧还蛋白（Rdox）（k = 3.0×10⁸ M⁻¹ s⁻¹）和氧化型铁氧化还原蛋白：NADP⁺还原酶（FNRox）还原至半醌水平（k = 5.5×10⁸ M⁻¹ s⁻¹）。发光黄素半醌还原Rdox的速度较慢（k = 1.3×10⁷ M⁻¹ s⁻¹），且与FNRox的反应无法检测到。吸收差光谱和差示圆二色光谱表明，在低离子强度（10 mM）下，Rdox和FNRox形成1:1复合物，而在较高离子强度（310 mM）下该复合物完全解离。发光黄素半醌还原游离态和复合态Rdox的表观二级速率常数相同。以5-dRf作为还原剂，研究了游离FNR半醌对Rdox（游离态和复合态）的还原以及复合物内电子转移。在I = 10 mM时，得到一级速率常数为2.0×10³ s⁻¹，这对应于复合物内从FNR半醌到Rdox的电子转移过程。还观察到游离FNR半醌与复合态Rdox之间发生二级反应（k = 5×10⁷ M⁻¹ s⁻¹）。在I = 310 mM时，未观察到这些反应，且FNR半醌与游离Rdox的反应为二级反应（k = 4×10⁶ M⁻¹ s⁻¹）。