铁氧化还原蛋白-铁氧化还原蛋白-NADP⁺还原酶复合物的还原动力学：激光闪光光解研究

Reduction kinetics of the ferredoxin-ferredoxin-NADP+ reductase complex: a laser flash photolysis study.

作者信息

Bhattachryya A K, Meyer T E, Tollin G

出版信息

Biochemistry. 1986 Aug 12;25(16):4655-61. doi: 10.1021/bi00364a030.

Abstract

The kinetics of reduction of spinach ferredoxin (Fd), ferredoxin-NADP+ reductase (FNR), and the Fd-FNR complex have been investigated by the laser flash photolysis technique. 5-Deazariboflavin semiquinone (5-dRf), generated in situ by laser flash photolysis under anaerobic conditions, rapidly reduced both oxidized Fd (Fdox) (k = 2 X 10(8) M-1 s-1) and oxidized FNR (FNRox) (K = 6.3 X 10(8) M-1 s-1) at low ionic strength (10 mM) at pH 7.0, leading to the formation of reduced Fd (Fdred) and FNR semiquinone (FNR.), respectively. At higher ionic strengths (310 and 460 mM), the rate constant for the reduction of the free Fdox increased about 3-fold (k = 6.7 X 10(8) M-1 s-1 at 310 mM and 6.4 X 10(8) M-1 s-1 at 460 mM). No change in the second-order rate constant for reduction of the free FNRox was observed at high ionic strength. At low ionic strength (10 mM), 5-dRf. reacted only with the FAD center of the preformed 1:1 Fdox-FNRox complex (k = 5.6 X 10(8) M-1 s-1), leading to the formation of FNR.. No direct reduction of Fdox in the complex was observed. No change in the kinetics occurred in the presence of excess NADP+. The second-order rate constant for reduction of Fdox by 5-dRf. in the presence of a stoichiometric amount of fully reduced FNR at low ionic strength was 7 X 10(6) M-1 s-1, i.e., about one-thirtieth the rate constant for reduction of free Fdox.(ABSTRACT TRUNCATED AT 250 WORDS)

摘要

通过激光闪光光解技术研究了菠菜铁氧还蛋白（Fd）、铁氧还蛋白-NADP⁺还原酶（FNR）以及Fd-FNR复合物的还原动力学。在厌氧条件下通过激光闪光光解原位生成的5-脱氮核黄素半醌（5-dRf），在pH 7.0、低离子强度（10 mM）时能快速还原氧化型Fd（Fdox）（k = 2×10⁸ M⁻¹ s⁻¹）和氧化型FNR（FNRox）（K = 6.3×10⁸ M⁻¹ s⁻¹），分别生成还原型Fd（Fdred）和FNR半醌（FNR.）。在较高离子强度（310 mM和460 mM）下，游离Fdox还原的速率常数增加了约3倍（310 mM时k = 6.7×10⁸ M⁻¹ s⁻¹，460 mM时k = 6.4×10⁸ M⁻¹ s⁻¹）。在高离子强度下未观察到游离FNRox还原的二级速率常数有变化。在低离子强度（10 mM）时，5-dRf.仅与预先形成的1:1 Fdox-FNRox复合物的FAD中心反应（k = 5.6×10⁸ M⁻¹ s⁻¹），生成FNR.。未观察到复合物中Fdox的直接还原。在过量NADP⁺存在下动力学没有变化。在低离子强度下，当存在化学计量的完全还原型FNR时，5-dRf.还原Fdox的二级速率常数为7×10⁶ M⁻¹ s⁻¹，即约为游离Fdox还原速率常数的三十分之一。（摘要截短于250字）