COORDINATION OF SELENIUM TO MOLYBDENUM IN FORMATE DEHYDROGENASE-H FROM ESCHERICHIA-COLI

Formate dehydrogenase H from Escherichia coli contains multiple redox centers, which include a molybdopterin cofactor, an iron-sulfur center , and a selenocysteine residue (SeCys-140 in the polypeptide chain) th at 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 paramagnetic resonance (EPR) spe ctroscopy. Substitution of Se-77 for natural isotope abundance Se lead s to transformation of this signal, indicating a direct coordination o f Se with Mo. Mutant enzyme with cysteine substituted at position 140 for the selenocysteine residue has decreased catalytic activity and ex hibits a different EPR signal. Since determination of the Se content o f wild-type enzyme indicates approximate to 1 gram atom per mel, we co nclude that it is the Se atom of the SeCys-140 residue in the protein that is coordinated directly with Mo. The amino acid sequence flanking the selenocysteine residue in formate dehydrogenase H is similar to a conserved sequence found in several other prokaryotic molybdopterin-d ependent enzymes. In most of these other enzymes a cysteine residue, o r in a few cases a serine or a selenocysteine residue, occurs in the p osition corresponding to SeCys-140 of formate dehydrogenase H. By anal ogy with formate dehydrogenase H in these other enzymes, at least one of the ligands to Mo should be provided by an amino acid residue of th e protein. This ligand could be the Se of a selenocysteine residue, su lfur of a cysteine residue, or, in the case of a serine residue, oxyge n.