The O2-tolerant, NAD+-reducing soluble [NiFe] hydrogenase (SH) from Ralstonia eutropha H16, HoxHYFUI2, is a complex enzyme, harboring multiple redox cofactors: a [NiFe] active site, an electron relay of iron-sulfur clusters, and two noncovalently bound flavin mononucleotides (FMN). The interplay and functional role of these cofactors is so far not understood in detail. In the present study, the isolated HoxHY module was investigated, which represents the smallest active subcomplex of a [NiFe] hydrogenase. Direct electrochemical studies and solution assays showed that the as-isolated HoxHY is initially catalytically inactive, but after reductive activation at low potentials, exhibits both H2 oxidation and H+ reduction, consistent with the role of the SH in bidirectional catalysis. The overpotential relative to E(2H+/H2) is minimal, facilitating coupling of the closely spaced 2H+/H2 and NAD+/NADH half reactions in the SH. Methyl viologen reduction assays revealed that H2 oxidation by HoxHY is enhanced on addition of excess FMN, in line with results from optical spectroscopy which indicate that FMN is present at substoichiometric levels in as-isolated HoxHY. X-ray absorption spectroscopy suggested one 4Fe4S cluster in addition to the active site in HoxHY. FTIR investigations confirmed that the active site iron atom has a “standard” ligation, i.e., one CO and two cyanide ligands. At least two novel oxidized states were detected by FTIR, both of which could be reductively activated by artificial electron donors, such as dithionite, and by the native electron donor H2 in the presence of additional FMN. The flavin cofactor also appears to stabilize the active site, providing further evidence for its importance in HoxHY. All reduced states of the [NiFe] site previously identified for standard [NiFe] hydrogenases and for the native SH within living cells were detected in FTIR spectra of HoxHY with the exception of the intermediate Nia-C species. Electrochemical experiments show that incubation of active HoxHY with O2 at high potentials causes slow inactivation, but activity is recovered within seconds at potentials below –170 mV at 30 °C, even in the presence of 2 % O2. This behavior is consistent with the HoxHY moiety of the SH remaining active in the presence of O2 at the potential of the NAD+/NADH pool in vivo.