Thorneley R N, Deistung J
A.F.R.C.-I.P.S.R., Nitrogen Fixation Laboratory, University of Sussex, Brighton, U.K.
Biochem J. 1988 Jul 15;253(2):587-95. doi: 10.1042/bj2530587.
The kinetics of electron-transfer reactions involving flavodoxins from Klebsiella pneumoniae (KpFld), Azotobacter chroococcum (AcFld), Anacystis nidulans (AnFld) and Megasphaera elsdenii (MeFld), the free, MgADP-bound and MgATP-bound forms of the Fe protein component of nitrogenase from K. pneumoniae [Kp2, Kp2(MgADP)2 and Kp2(MgATP)2] and Na2S2O4 were studied by stopped-flow spectrophotometry. Kinetic evidence was obtained for the formation of binary protein complexes involving KpFldSQ (semiquinone) with either Kp2(MgADP)2 (KD = 49 microM) or Kp2(MgATP)2 (KD = 13 microM) but not with Kp2 (KD greater than 730 microM). The binding of 2MgATP or 2MgADP to Kp2 therefore not only shifts the midpoint potential (Em) of the [4Fe-4S] centre from -200 mV to -320 mV or -350 mV respectively but also changes the affinity of Kp2 for KpFldSQ. Thermodynamically unfavourable electron from Kp2(MgADP)2 and Kp2(MgATP)2 to KpFldSQ occurs within the protein complexes with k = 1.2 s-1 (delta E = -72 mV) and 0.5 s-1 (delta E = -120 mV) respectively. Although AcFldSQ is reduced by Kp2, Kp2(MgADP)2 and Kp2(MgATP)2 (k = 8 x 10(3), 2.4 x 10(3) and 9 x 10(2) M-1.s-1 respectively), protein-complex formation is weak in each case (KD greater than 700 microM). Electron transfer in the physiologically important and thermodynamically favourable direction from Kp2FldHQ (hydroquinone) and AcFldHQ to Kp2ox.(MgADP)2 (the state of Kp2 that accepts electrons from FldHQ in the catalytic cycle of nitrogenase) is rapid (k greater than 10(6) M-1.s-1). The second-order rate constants for the reduction of KpFldSQ, AcFldSQ, AnFldSQ and MeFldSQ by SO2.- (active reductant formed by the predissociation of S2O4(2-) ion) exhibited the linear free-energy relationship predicted by the Marcus theory of electron transfer.
利用停流分光光度法研究了涉及肺炎克雷伯菌黄素氧还蛋白（KpFld）、褐球固氮菌黄素氧还蛋白（AcFld）、集胞藻黄素氧还蛋白（AnFld）和埃氏巨球型菌黄素氧还蛋白（MeFld）的电子转移反应动力学，以及肺炎克雷伯菌固氮酶铁蛋白组分的游离形式、MgADP结合形式和MgATP结合形式[Kp2、Kp2(MgADP)2和Kp2(MgATP)2]与连二亚硫酸钠（Na2S2O4）的反应动力学。动力学证据表明，KpFldSQ（半醌）与Kp2(MgADP)2（解离常数KD = 49 μM）或Kp2(MgATP)2（KD = 13 μM）形成二元蛋白质复合物，但不与Kp2形成（KD大于730 μM）。因此，2MgATP或2MgADP与Kp2的结合不仅将[4Fe-4S]中心的中点电位（Em）分别从-200 mV变为-320 mV或-350 mV，还改变了Kp2对KpFldSQ的亲和力。在蛋白质复合物中，从Kp2(MgADP)2和Kp2(MgATP)2到KpFldSQ的热力学不利电子转移分别以k = 1.2 s-1（ΔE = -72 mV）和0.5 s-1（ΔE = -120 mV）的速率发生。尽管AcFldSQ可被Kp2、Kp2(MgADP)2和Kp2(MgATP)2还原（速率常数分别为8×10³、2.4×10³和9×10² M⁻¹·s⁻¹），但每种情况下蛋白质复合物的形成都很弱（KD大于700 μM）。在生理上重要且热力学有利的方向上，从Kp2FldHQ（对苯二酚）和AcFldHQ到Kp2ox.(MgADP)2（在固氮酶催化循环中从FldHQ接受电子的Kp2状态）的电子转移很快（k大于10⁶ M⁻¹·s⁻¹）。SO₂⁻（由S₂O₄²⁻离子预解离形成的活性还原剂）还原KpFldSQ、AcFldSQ、AnFldSQ和MeFldSQ的二级速率常数呈现出马库斯电子转移理论预测的线性自由能关系。
