Abstract
The NAD+-reducing soluble hydrogenase (SH) from Ralstonia eutropha H16 catalyzes the H2-driven reduction of NAD+, as well as reverse electron transfer from NADH to H+, in the presence of O2. It comprises six subunits, HoxHYFUI2, and incorporates a [NiFe] H+/H2 cycling catalytic centre, two non-covalently bound flavin mononucleotide (FMN) groups and an iron-sulfur cluster relay for electron transfer. This study provides the first characterization of the diaphorase sub-complex made up of HoxF and HoxU. Sequence comparisons with the closely related peripheral subunits of Complex I in combination with UV/Vis spectroscopy and the quantification of the metal and FMN content revealed that HoxFU accommodates a [2Fe2S] cluster, FMN and a series of [4Fe4S] clusters. Protein film electrochemistry (PFE) experiments show clear electrocatalytic activity for both NAD+ reduction and NADH oxidation with minimal overpotential relative to the potential of the NAD+/NADH couple. Michaelis-Menten constants of 56 µM and 197 µM were determined for NADH and NAD+, respectively. Catalysis in both directions is product inhibited with K I values of around 0.2 mM. In PFE experiments, the electrocatalytic current was unaffected by O2, however in aerobic solution assays, a moderate superoxide production rate of 54 nmol per mg of protein was observed, meaning that the formation of reactive oxygen species (ROS) observed for the native SH can be attributed mainly to HoxFU. The results are discussed in terms of their implications for aerobic functioning of the SH and possible control mechanism for the direction of catalysis.
Highlights
One obvious strategy of evolution is the recurrent use of conserved protein domains or even whole catalytic modules in different contexts allowing the connection of previously unlinked catalytic functions
The subcomplex is catalytically active in transferring electrons from NADH to benzyl viologen, and exhibits electrocatalytic NADH oxidation and NAD+ reduction on a graphite electrode
We have been able to examine the catalytic bias of the NAD+/ NADH site, its ability to function in the presence of O2, and its production of reactive oxygen species (ROS) under aerobic conditions
Summary
One obvious strategy of evolution is the recurrent use of conserved protein domains or even whole catalytic modules in different contexts allowing the connection of previously unlinked catalytic functions. The NADH dehydrogenase/diaphorase module of Complex I is part of NAD+reducing formate dehydrogenases, and hydrogenases [2,3] We concentrate on the functional characterization of the NADH dehydrogenase module of the NAD+reducing soluble [NiFe]-hydrogenase (SH) from the Knallgas bacterium Ralstonia eutropha H16 [4,5,6,7,8]. The SH belongs to a subclass of ‘‘bidirectional’’ [NiFe]-hydrogenases and provides cells with reducing equivalents in the form of NADH generated from H2 oxidation. The SH may function as an electron valve in vivo under conditions of excessive reductant supply, coupling NADH oxidation to H+ reduction [9], as proposed for the cyanobacterial bidirectional hydrogenases [10]. The SH belongs to a group of ‘O2 tolerant’ [NiFe]-hydrogenases including the membrane-bound hydrogenase from R. eutropha and the Hyd 1 enzymes of Aquifex aeolicus, and Escherichia coli [11,12,13]
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