Seo Daisuke, Soeta Takahiro, Sakurai Hidehiro, Sétif Pierre, Sakurai Takeshi
Division of Material Science, Graduate School of Natural Science and Technology, Kanazawa University, Kakuma, Kanazawa, Ishikawa 920-1192, Japan.
Division of Material Science, Graduate School of Natural Science and Technology, Kanazawa University, Kakuma, Kanazawa, Ishikawa 920-1192, Japan.
Biochim Biophys Acta. 2016 Jun;1857(6):678-87. doi: 10.1016/j.bbabio.2016.03.005. Epub 2016 Mar 8.
Ferredoxin-NADP(+) oxidoreductase ([EC1.18.1.2], FNR) from Bacillus subtilis (BsFNR) is a homodimeric flavoprotein sharing structural homology with bacterial NADPH-thioredoxin reductase. Pre-steady-state kinetics of the reactions of BsFNR with NADP(+), NADPH, NADPD (deuterated form) and B. subtilis ferredoxin (BsFd) using stopped-flow spectrophotometry were studied. Mixing BsFNR with NADP(+) and NADPH yielded two types of charge-transfer (CT) complexes, oxidized FNR (FNR(ox))-NADPH and reduced FNR (FNR(red))-NADP(+), both having CT absorption bands centered at approximately 600n m. After mixing BsFNR(ox) with about a 10-fold molar excess of NADPH (forward reaction), BsFNR was almost completely reduced at equilibrium. When BsFNR(red) was mixed with NADP(+), the amount of BsFNR(ox) increased with increasing NADP(+) concentration, but BsFNR(red) remained 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)). Mixing BsFd(red) with BsFNR(ox) induced 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(-1) at 10°C, which increased as BsFd(red) increased from 40 to 120 μM. The reduction rate of BsFNR(ox) by BsFd(red) was markedly decreased by premixing BsFNR(ox) with BsFd(ox), indicating that the dissociation of BsFd(ox) from BsFNR(sq) is rate-limiting in the reaction. The characteristics of the BsFNR reactions with NADP(+)/NADPH were compared with those of other types of FNRs.
来自枯草芽孢杆菌的铁氧化还原蛋白 - NADP(+)氧化还原酶（[EC1.18.1.2]，FNR，即BsFNR）是一种同二聚体黄素蛋白，与细菌NADPH - 硫氧还蛋白还原酶具有结构同源性。利用停流分光光度法研究了BsFNR与NADP(+)、NADPH、NADPD（氘代形式）以及枯草芽孢杆菌铁氧化还原蛋白（BsFd）反应的预稳态动力学。将BsFNR与NADP(+)和NADPH混合产生了两种类型的电荷转移（CT）复合物，即氧化型FNR（FNR(ox)） - NADPH和还原型FNR（FNR(red)） - NADP(+)，两者都具有以约600nm为中心的CT吸收带。将BsFNR(ox)与大约10倍摩尔过量的NADPH混合（正向反应）后，BsFNR在平衡时几乎完全被还原。当将BsFNR(red)与NADP(+)混合时，BsFNR(ox)的量随着NADP(+)浓度的增加而增加，但即使在大约50倍摩尔过量的NADP(+)存在下，BsFNR(red)在平衡时仍为主要形式。在两个方向上，氢化物转移都是速率决定步骤，其中正向反应速率常数（约500 s⁻¹）远高于逆向反应速率常数（<10 s⁻¹）。将BsFd(red)与BsFNR(ox)混合会诱导快速形成中性半醌形式。这个过程在1毫秒内几乎完成。随后，中性半醌形式在10°C下以50至70 s⁻¹的表观速率常数还原为对苯二酚形式，该速率常数随着BsFd(red)从40μM增加到120μM而增加。通过将BsFNR(ox)与BsFd(ox)预混合，BsFd(red)对BsFNR(ox)的还原速率显著降低，这表明BsFd(ox)从BsFNR(sq)的解离是该反应的限速步骤。将BsFNR与NADP(+)/NADPH反应的特性与其他类型的FNRs进行了比较。
