Spectroscopic and biochemical insight into an electron-bifurcating [FeFe] hydrogenase
Spectroscopic and biochemical insight into an electron-bifurcating [FeFe] hydrogenase
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DOI：
10.1007/s00775-019-01747-1
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发表时间：
2020-02-01
影响因子：
3
通讯作者：
Ogata, Hideaki
中科院分区：
文献类型：
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作者：
Chongdar, Nipa;Pawlak, Krzysztof;Ogata, Hideaki
来自Thermotoga maritima(TM)的异三聚体电子分叉[FeFe]氢酶(HydABC)将二氢烟酰胺腺嘌呤二核苷酸(NADH)对质子(H+)的能量还原(增量G(0)约为18kJ摩尔(-1))与还原铁还蛋白(Fd(Red))对H+的能量还原(增量G(0)约为-16kJ摩尔(-1))偶联。HydABC发挥作用的具体机制尚不清楚。在本研究中，我们描述了在大肠杆菌中重组生产的TmHydABC的生化和光谱特征，并用合成的双铁辅因子人工成熟。我们发现，在氧化铁还蛋白(FD(Ox))存在下，TmHydABC催化烟酰胺腺嘌呤二核苷酸(NAD(+))依赖于氢(H-2)还原的速率接近17mU·mol·NADH·min(-1)mg(-1)。我们的数据表明，酶中只有一个黄素，不太可能是电子分叉的位置。FTIR和EPR光谱以及FTIR光谱电化学表明，TmHydABC中H-2转化的活性中心H-簇与典型的[FeFe]氢酶基本相同，也很可能不是电子分叉的位置。结合目前关于[FeFe]氢酶电子分叉机制的假说，讨论了这些结果的意义。总体而言，这些结果提供了对电子分叉机制的洞察，并为进一步研究这类令人着迷的[FeFe]氢酶提供了一个明确的系统。图形摘要
The heterotrimeric electron-bifurcating [FeFe] hydrogenase (HydABC) from Thermotoga maritima (Tm) couples the endergonic reduction of protons (H+) by dihydronicotinamide adenine dinucleotide (NADH) ( increment G(0) approximate to 18 kJ mol(-1)) to the exergonic reduction of H+ by reduced ferredoxin (Fd(red)) ( increment G(0) approximate to - 16 kJ mol(-1)). The specific mechanism by which HydABC functions is not understood. In the current study, we describe the biochemical and spectroscopic characterization of TmHydABC recombinantly produced in Escherichia coli and artificially maturated with a synthetic diiron cofactor. We found that TmHydABC catalyzed the hydrogen (H-2)-dependent reduction of nicotinamide adenine dinucleotide (NAD(+)) in the presence of oxidized ferredoxin (Fd(ox)) at a rate of approximate to 17 mu mol NADH min(-1) mg(-1). Our data suggest that only one flavin is present in the enzyme and is not likely to be the site of electron bifurcation. FTIR and EPR spectroscopy, as well as FTIR spectroelectrochemistry, demonstrated that the active site for H-2 conversion, the H-cluster, in TmHydABC behaves essentially the same as in prototypical [FeFe] hydrogenases, and is most likely also not the site of electron bifurcation. The implications of these results are discussed with respect to the current hypotheses on the electron bifurcation mechanism of [FeFe] hydrogenases. Overall, the results provide insight into the electron-bifurcating mechanism and present a well-defined system for further investigations of this fascinating class of [FeFe] hydrogenases. Graphic abstract