Department of Biochemistry and the Biophysics Graduate Program, University of California, Riverside, USA.
Biosciences Center, National Renewable Energy Laboratory, Golden, Colorado, USA.
J Biol Chem. 2023 Dec;299(12):105403. doi: 10.1016/j.jbc.2023.105403. Epub 2023 Oct 29.
We have investigated the kinetics of NAD-dependent NADPH:ferredoxin oxidoreductase (NfnI), a bifurcating transhydrogenase that takes two electron pairs from NADPH to reduce two ferredoxins and one NAD through successive bifurcation events. NADPH reduction takes place at the bifurcating FAD of NfnI's large subunit, with high-potential electrons transferred to the [2Fe-2S] cluster and S-FADH of the small subunit, ultimately on to NAD; low-potential electrons are transferred to two [4Fe-4S] clusters of the large subunit and on to ferredoxin. Reduction of NfnI by NADPH goes to completion only at higher pH, with a limiting k of 36 ± 1.6 s and apparent K of 5 ± 1.2 μM. Reduction of one of the [4Fe-4S] clusters of NfnI occurs within a second, indicating that in the absence of NAD, the system can bifurcate and generate low-potential electrons without NAD. When enzyme is reduced by NADPH in the absence of NAD but the presence of ferredoxin, up to three equivalents of ferredoxin become reduced, although the reaction is considerably slower than seen during steady-state turnover. Bifurcation appears to be limited by transfer of the first, high-potential electron into the high-potential pathway. Ferredoxin reduction without NAD demonstrates that electron bifurcation is an intrinsic property of the bifurcating FAD and is not dependent on the simultaneous presence of NAD and ferredoxin. The tight coupling between NAD and ferredoxin reduction observed under multiple-turnover conditions is instead simply due to the need to remove reducing equivalents from the high-potential electron pathway under multiple-turnover conditions.
我们研究了 NAD 依赖性 NADPH：铁氧还蛋白氧化还原酶（NfnI）的动力学，NfnI 是一种分叉的转氢酶，通过连续的分叉事件从 NADPH 中获取两个电子对，还原两个铁氧还蛋白和一个 NAD。NADPH 的还原发生在 NfnI 大亚基的分叉 FAD 上，高势能电子被转移到[2Fe-2S]簇和小亚基的 S-FADH，最终转移到 NAD；低势能电子被转移到大亚基的两个[4Fe-4S]簇，并转移到铁氧还蛋白上。只有在较高的 pH 值下，NfnI 才能被 NADPH 完全还原，限速常数为 36±1.6 s-1，表观 K 为 5±1.2 μM。NfnI 中一个[4Fe-4S]簇的还原在一秒内发生，表明在没有 NAD 的情况下，该系统可以分叉并产生不需要 NAD 的低势能电子。当酶在没有 NAD 的情况下被 NADPH 还原，但存在铁氧还蛋白时，多达三个当量的铁氧还蛋白被还原，尽管反应速度比稳态周转时慢得多。分叉似乎受到第一个高势能电子进入高势能途径的转移限制。没有 NAD 时铁氧还蛋白的还原表明，电子分叉是分叉 FAD 的固有特性，不依赖于 NAD 和铁氧还蛋白的同时存在。在多次周转条件下观察到的 NAD 和铁氧还蛋白还原之间的紧密偶联，仅仅是因为在多次周转条件下需要从高势能电子途径中去除还原当量。
