Abstract

The heterotrimeric electron-bifurcating [FeFe] hydrogenase (HydABC) from Thermotoga maritima (Tm) couples the endergonic reduction of protons (H⁺) by dihydronicotinamide adenine dinucleotide (NADH) (∆G⁰ ≈ 18 kJ mol⁻¹) to the exergonic reduction of H⁺ by reduced ferredoxin (Fd_red) (∆G⁰ ≈ − 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 (H₂)-dependent reduction of nicotinamide adenine dinucleotide (NAD⁺) in the presence of oxidized ferredoxin (Fd_ox) at a rate of ≈17 μmol 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 H₂ 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

中文翻译：

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电子分叉[FeFe]氢化酶的光谱学和生化分析。

抽象的

异源电子分叉从[FEFE]氢化（HydABC）海栖热袍（的Tm）耦合质子的吸能还原（H ⁺通过dihydronicotinamide腺嘌呤二核苷酸（NADH））（Δ G ^ ⁰  ≈18千焦耳摩尔^-1）到放能减少H的⁺由减少的铁氧还蛋白（FD_红）（Δ G ^ ⁰  ≈ - 16千焦摩尔^-1）。尚不清楚HydABC发挥作用的具体机制。在当前的研究中，我们描述了在大肠杆菌中重组产生的Tm HydABC的生化和光谱表征并与合成的二铁辅因子人工成熟。我们发现，Tm HydABC在氧化铁氧还蛋白（Fd _ox）的存在下以≈17μmolNADH min ^-1  mg ^-1的速率催化烟酰胺腺嘌呤二核苷酸（NAD ⁺）的氢（H ₂）依赖性还原。我们的数据表明，酶中仅存在一种黄素，并且不太可能是电子分叉的位点。FTIR和EPR光谱以及FTIR光谱电化学表明，Tm中H ₂转化的活性位点H簇HydABC的行为与典型的[FeFe]氢化酶基本相同，并且很可能不是电子分叉的位点。关于[FeFe]氢化酶的电子分叉机制的当前假设，讨论了这些结果的含义。总体而言，结果为深入了解电子分叉机制提供了一个清晰的系统，可用于进一步研究这种引人入胜的[FeFe]氢化酶类别。

图形摘要