Spectroscopic and biochemical insight into an electron-bifurcating [FeFe] hydrogenase

Nipa Chongdar,Olaf Rüdiger,Wolfgang Lubitz,Krzysztof Pawlak,Hideaki Ogata,James A Birrell,Edward J Reijerse,Patricia Rodríguez-Maciá

doi:10.1007/s00775-019-01747-1

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 (Fdred) (∆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 (Fdox) 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

Highlights

Abbreviations NADH Dihydronicotinamide adenine dinucleotide NAD+ Nicotinamide adenine dinucleotide Fdox Oxidized ferredoxin Fdred Reduced ferredoxin ADT 2-azapropane 1,3-dithiolate flavin mononucleotide (FMN) Flavin mononucleotide FTIR Fourier transform infrared electron paramagnetic resonance (EPR) Electron paramagnetic resonance flavin adenine dinucleotide (FAD) Flavin adenine dinucleotide continuous wave (CW) Continuous wave open-circuit potential (OCP) Open-circuit potential proton coupled electron transfer (PCET) Proton-coupled electron transfer FBEB Flavin-based electron bifurcation
Iron quantification of apo-TmHydABC and apo-TmHydA indicated the presence of 35 ± 2 and ± 1 moles of iron per mole of protein, respectively, in good agreement with the expected values (36 for apo-TmHydABC and for apo-TmHydA) based on the number of iron–sulfur (FeS) clusters, and on the previously reported data on TmHydABC and TmHydA isolated from the native organism [26]
It can be concluded that apoTmHydABC and apo-TmHydA contain both [2Fe–2S] and [4Fe–4S] clusters essentially identical to that of the proteins purified from the native organism

Summary

Introduction

Abbreviations NADH Dihydronicotinamide adenine dinucleotide NAD+ Nicotinamide adenine dinucleotide Fdox Oxidized ferredoxin Fdred Reduced ferredoxin ADT 2-azapropane 1,3-dithiolate FMN Flavin mononucleotide FTIR Fourier transform infrared EPR Electron paramagnetic resonance FAD Flavin adenine dinucleotide CW Continuous wave OCP Open-circuit potential PCET Proton-coupled electron transfer FBEB Flavin-based electron bifurcation. Subsequent investigations showed that in a variety of reactions in anaerobic metabolism, electron bifurcation is involved [20], among them: coupling of the endergonic oxidation of NADH (E0′ = − 320 mV) to the exergonic oxidation of reduced ferredoxin (Fdred) (E0′ ≈ − 450 mV) to reduce. This reaction is essential in many organisms as various metabolic steps only yield enough energy to reduce nicotinamide adenine dinucleotide (NAD+), but NADH is not capable of reducing protons. Understanding the metabolism of these organisms may be important for medical applications

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