Catalytic Properties of the Isolated Diaphorase Fragment of the NAD+-Reducing [NiFe]-Hydrogenase from Ralstonia eutropha

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.

The oxygen‐tolerant and NAD+‐dependent formate dehydrogenase from Rhodobacter capsulatus is able to catalyze the reduction of CO2 to formate

“…This is consistent with a previous report on the soluble hydrogenase from Ra. eutropha H16, where substrate inhibition with concentrations of 200 lM NADH and higher was also observed [26].…”

“…eutropha shows high identities between the HoxF and HoxU subunits and R. capsulatus FdsG and FdsB. HoxF and HoxU were recently shown to form an efficient diaphorase module [26]. To determine the kinetic constants of FdsGB for NAD + reduction and NADH oxidation, we purified the FdsGB heteromer after separate expression of fdsG and fdsB in E. coli (Fig.…”

An Innovative Cloning Platform Enables Large-Scale Production and Maturation of an Oxygen-Tolerant [NiFe]-Hydrogenase from Cupriavidus necator in Escherichia coli

“…A cytoplasmic bidirectional hydrogenase (soluble hydrogenase, SH), the target of this project, links H 2 oxidation to the reduction of NAD + , thereby forming reducing equivalents for respiration and CO 2 fixation. The SH consists of six subunits and can be subdivided structurally into two distinct modules: A hydrogenase moiety HoxYH, accompanied by an NADH:oxidoreductase (diaphorase) module, termed HoxFU, with sequence similarities to the peripheral subunits of mitochondrial Complex I [16]. The hexameric structure is completed by a homodimer of HoxI, a small subunit bound to the diaphorase module, which serves as an NADPH activation site of the oxidized enzyme [17].…”

4 Oxygen-tolerant hydrogenases and their biotechnological potential

“…Based on amino acid sequence similarities with the closely related peripheral subunits of Complex I, the NADH:acceptor oxidoreductase module (HoxFU) accommodates three [4Fe4S] clusters, one [2Fe2S] cluster and one FMN molecule (designated FMN-b, [64,65]). FMN-b is a part of the NAD(H) binding pocket and acts as a communicator between the two-electron accommodating cofactor NADH and the Fe-S clusters, which normally transfer only one electron at a time [66]. FMN-b is a part of the NAD(H) binding pocket and acts as a communicator between the two-electron accommodating cofactor NADH and the Fe-S clusters, which normally transfer only one electron at a time [66].…”