Formate dehydrogenase H from Escherichia col contains multiple redox centers, which include a molybdopterin cofactor, an iron-sulfur center, and a selenocysteine residue (SeCys-140 in the polypeptide chain) that is essential for catalytic activity. Here we show that addition of formate to the native enzyme induces a signal typical of Mo(V) species. This signal is detected by electron pm etc resonance (EPR) spectroscopy. Substitution of 77Se for natural isotope abundance Se leads to transformation of this signal, indicating a direct coordination of Se with Mo. Mutant enzyme with cysteine substituted at position 140 for the selenocysteine residue has decreased catalytic activity and exhibits a different EPR signal. Since deternation of the Se content of wild-type enzyme indicates 1 gram atom per mol, we conclude that it is the Se atom of the SeCys-140 residue in the protein that is coordinated directly with Mo. The amino acd sequence flanking the selenocysteine residue in formate dehydrogenase H is s r to a conserved sequence found in several other prokaryotic molybdopterin-dependent enzymes. In most of these other enzymes a cysteine residue, or in a few cases a serine or a selenocysteine residue, occurs in the position corresonding to SeCys-140 of formate dehydrogeme H. By analogy with formate dehydrogenase H in these other enzymes, at least one of the ilgands to Mo should be provided by an amino acid residue of the protein. This igand could be the Se of a selenocysteine residue, sulfur of a cysteine residue, or, in the case of a serine residue, oxygen
