Infrared-spectroscopic studies on the [NiFe]-hydrogenase of Chromatium vinosum-enriched in 15N or 13C, as well as chemical analyses, show that this enzyme contains three non-exchangeable, intrinsic, diatomic molecules as ligands to the active site, one carbon monoxide molecule and two cyanide groups. The results form an explanation for the three non-protein ligands to iron detected in the crystal structure of the Desulfovibrio gigas hydrogenase (Volbeda, A., Garcin, E., Piras, C., De Lacey, A. I., Fernandez, V. M., Hatchikian, E. C., Frey, M., and Fontecilla-Camps, J. C. (1996) J. Am. Chem. Soc. 118, 12989-12996) and for the low spin character of the lone ferrous iron ion observed with Mössbauer spectroscopy (Surerus, K. K., Chen, M., Van der Zwaan, W., Rusnak, F. M., Kolk, M. , Duin, E. C., Albracht, S. P. J., and Münck, E. (1994) Biochemistry 33, 4980-4993). The results do not support the notion, based upon studies of Desulfovibrio vulgaris [NiFe]-hydrogenase (Higuchi, Y., Yagi, T., and Noritake, Y. (1997) Structure 5, 1671-1680), that SO is a ligand to the active site. The occurrence of both cyanide and carbon monoxide as intrinsic constituents of a prosthetic group is unprecedented in biology.