Pre-steady-state kinetic studies of redox reactions catalysed by Bacillus subtilis ferredoxin-NADP+ oxidoreductase with NADP+/NADPH and ferredoxin
Pre-steady-state kinetic studies of redox reactions catalysed by Bacillus subtilis ferredoxin-NADP+ oxidoreductase with NADP+/NADPH and ferredoxin
复制标题
枯草芽孢杆菌铁氧还蛋白-NADP氧化还原酶与NADP /NADPH和铁氧还蛋白催化氧化还原反应的前稳态动力学研究
DOI：
10.1016/j.bbabio.2016.03.005
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发表时间：
2016
期刊：
影响因子：
--
通讯作者：
Takeshi Sakurai
中科院分区：
文献类型：
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作者：
Daisuke Seo;Takahiro Soeta;Hidehiro Sakurai;Pierre Setif;Takeshi Sakurai
枯草芽孢杆菌（Bacillussubtilis，BsFNR）的铁氧还蛋白-NADP+氧化还原酶（Ferredoxin-NADP+oxidoreductase，[EC1.18.1.2]，FNR）是一种与细菌NADPH-硫氧还蛋白还原酶具有结构同源性的同源二聚体黄素蛋白。研究了BsFNR与NADP+、NADPH、NADPH（氘代）和B.采用停流分光光度法对枯草杆菌铁氧还蛋白（BsFd）进行了测定。将BsFNR与NADP+和NADPH混合，得到两种类型的电荷转移（CT）复合物，氧化的FNR（FNRox）-NADPH和还原的FNR（FNRred）-NADP+，两者都具有中心位于约600 nm的CT吸收带。在将BsFNR与约10倍摩尔过量的NADPH混合后（正向反应），BsFNR在平衡时几乎完全还原。当BsFNRred与NADP+混合时，BsFNRox的量随NADP+浓度的增加而增加，但即使在约50倍摩尔过量的NADP+下，BsFNRred仍保持为平衡的主要物种。在这两个方向上，脱附转移是速率决定步骤，其中正向速率常数（~ 500 s-1）远高于反向速率常数（< 10 s-1）。混合BsFdredge与BsFNRox诱导快速形成中性半醌形式。这个过程几乎在1 ms内完成。随后，中性半醌形式被还原为氢醌形式，在10 °C下的表观速率常数为50至70 s− 1，随着BsFdredge从40增加到120 μM而增加。将BsFNRox与BsFdox预混后，BsFdredge对BsFNRox的还原速率明显降低，表明BsFdox与BsFNRsq 2的解离是反应的限速过程。比较了BsFNR与NADP+/NADPH反应的特点。
Ferredoxin-NADP+oxidoreductase ([EC1.18.1.2], FNR) fromBacillus subtilis(BsFNR) is a homodimeric flavoprotein sharing structural homology with bacterial NADPH-thioredoxin reductase. Pre-steady-state kinetics of the reactions ofBsFNR with NADP+, NADPH, NADPD (deuterated form) andB. subtilisferredoxin (BsFd) using stopped-flow spectrophotometry were studied. MixingBsFNR with NADP+and NADPH yielded two types of charge-transfer (CT) complexes, oxidized FNR (FNRox)-NADPH and reduced FNR (FNRred)-NADP+, both having CT absorption bands centered at approximately 600 nm. After mixingBsFNRoxwith about a 10-fold molar excess of NADPH (forward reaction),BsFNR was almost completely reduced at equilibrium. WhenBsFNRredwas mixed with NADP+, the amount ofBsFNRoxincreased with increasing NADP+concentration, butBsFNRredremained as the major species at equilibrium even with about 50-fold molar excess NADP+. In both directions, the hydride-transfer was the rate-determining step, where the forward direction rate constant (~ 500 s− 1) was much higher than the reverse one (< 10 s− 1). MixingBsFdredwithBsFNRoxinduced rapid formation of a neutral semiquinone form. This process was almost completed within 1 ms. Subsequently the neutral semiquinone form was reduced to the hydroquinone form with an apparent rate constant of 50 to 70 s− 1at 10 °C, which increased asBsFdredincreased from 40 to 120 μM. The reduction rate ofBsFNRoxbyBsFdredwas markedly decreased by premixingBsFNRoxwithBsFdox, indicating that the dissociation ofBsFdoxfromBsFNRsqis rate-limiting in the reaction. The characteristics of theBsFNR reactions with NADP+/NADPH were compared with those of other types of FNRs.