The iron-sulfur cluster-free hydrogenase (Hmd) from methanogenic archaea harbors an iron-containing, light-sensitive cofactor of still unknown structure as prosthetic group. The enzyme is reversibly inhibited by CO and cyanide and is EPR silent. We report here on Mössbauer spectra of the (57)Fe-labeled enzyme and of the isolated cofactor. The spectrum of the holoenzyme measured at 80 K revealed a doublet peak with an isomer shift delta = 0.06 mm.s(-)(1) and a quadrupole splitting of DeltaE(Q) = 0.65 mm.s(-)(1) (at pH 8.0). The signal intensity corresponded to the enzyme concentration assuming 1 Fe per mol active site. Upon addition of CO or cyanide to the enzyme, the isomer shift decreased to -0.03 mm.s(-)(1) and -0.00(1) mm.s(-)(1), and the quadrupole splitting increased to 1.38 mm.s(-)(1) and 1.75 mm.s(-)(1), respectively. The three spectra could be perfectly simulated assuming the presence of only one type of iron in Hmd. The low isomer shift is characteristic for Fe in a low oxidation state (0, +1, +2). When the spectra of the holoenzyme and of the CO- or cyanide-inhibited enzyme were measured at 4 K in a magnetic field of 4 and 7 T, the spectra obtained could be simulated assuming the presence of only the external magnetic field, which excludes that the iron in the active site of Hmd is Fe(I), high-spin Fe(0), or high-spin Fe(II). Mössbauer spectra of the isolated Hmd cofactor are also reported.