The Nonphysiological Reductant Sodium Dithionite and [FeFe] Hydrogenase: Influence on the Enzyme Mechanism.

Maria Alessandra Martini,Olaf Rüdiger,Nina Breuer,Serena Debeer,Birgit Nöring,James A Birrell,Patricia Rodríguez-Maciá

doi:10.1021/jacs.1c07322

Abstract

[FeFe] hydrogenases are highly active enzymes for interconverting protons and electrons with hydrogen (H2). Their active site H-cluster is formed of a canonical [4Fe-4S] cluster ([4Fe-4S]H) covalently attached to a unique [2Fe] subcluster ([2Fe]H), where both sites are redox active. Heterolytic splitting and formation of H2 takes place at [2Fe]H, while [4Fe-4S]H stores electrons. The detailed catalytic mechanism of these enzymes is under intense investigation, with two dominant models existing in the literature. In one model, an alternative form of the active oxidized state Hox, named HoxH, which forms at low pH in the presence of the nonphysiological reductant sodium dithionite (NaDT), is believed to play a crucial role. HoxH was previously suggested to have a protonated [4Fe-4S]H. Here, we show that HoxH forms by simple addition of sodium sulfite (Na2SO3, the dominant oxidation product of NaDT) at low pH. The low pH requirement indicates that sulfur dioxide (SO2) is the species involved. Spectroscopy supports binding at or near [4Fe-4S]H, causing its redox potential to increase by ∼60 mV. This potential shift detunes the redox potentials of the subclusters of the H-cluster, lowering activity, as shown in protein film electrochemistry (PFE). Together, these results indicate that HoxH and its one-electron reduced counterpart Hred′H are artifacts of using a nonphysiological reductant, and not crucial catalytic intermediates. We propose renaming these states as the “dithionite (DT) inhibited” states Hox-DTi and Hred-DTi. The broader potential implications of using a nonphysiological reductant in spectroscopic and mechanistic studies of enzymes are highlighted.

Highlights

ABSTRACT: [FeFe] hydrogenases are highly active enzymes for interconverting protons and electrons with hydrogen (H2)
In the absence of structural models supported by experimental data, computational chemistry has played an important role in proposing likely structures of the active site in the catalytic intermediates based on spectroscopic data
The effect of this on catalysis was investigated via protein film electrochemistry (PFE), showing that binding of SO2 has an inhibitory effect on both H+ reduction and H2 oxidation activity of [FeFe] hydrogenases

Summary

■ INTRODUCTION

[FeFe] hydrogenases are highly active metalloenzymes that catalyze the reversible reduction of protons to molecular hydrogen.[1,2] Their active site, the H-cluster, comprises a unique diiron subcluster ([2Fe]H) and a canonical [4Fe-4S] subcluster ([4Fe-4S]H), covalently linked by a cysteine thiolate[3,4] (Figure 1 A and B). Based on the ratios of the Hox/ Hred and HoxH/Hred′H states under H2, binding of SO2 causes the redox potential of [4Fe-4S]H to increase by ∼60 mV The effect of this on catalysis was investigated via protein film electrochemistry (PFE), showing that binding of SO2 has an inhibitory effect on both H+ reduction and H2 oxidation activity of [FeFe] hydrogenases. Together, these results reveal that the so-called HoxH and Hred′H states are not related to protonation events at the [4Fe-4S]H subcluster of the H-cluster, but are instead artifacts generated by oxidized. These findings highlight the importance of carefully considering the possible side-reactions of NaDT and its oxidation products when choosing to use this reducing agent with metalloenzymes, iron−sulfur enzymes

■ RESULTS

■ CONCLUSIONS

■ ACKNOWLEDGMENTS

■ REFERENCES