Soluble NAD-reducing [NiFe]-hydrogenase (SH) from Ralstonia eutropha (formerly Alcaligenes eutrophus) has an infrared spectrum with one strong band at 1956 cm−1 and four weak bands at 2098, 2088, 2081 and 2071 cm−1 in the 2150–1850 cm−1 spectral region. Other [NiFe]-hydrogenases only show one strong and two weak bands in this region, attributable to the NiFe(CN)2(CO) active site. The position of these three bands is highly sensitive to redox changes of the active site. In contrast, reduction of the SH resulted in a shift to lower frequencies of the 2098 cm−1 band only. These and other properties prompted us to propose the presence of a Ni(CN)Fe(CN)3(CO) active site.
