Resonance Raman characterization of the heme cofactor in cystathionine beta-synthase. Identification of the Fe-S(Cys) vibration in the six-coordinatelow-spin heme

Human cystathionine beta -synthase (CBS) is an essential enzyme for the rem oval of the toxic metabolite homocysteine. Heme and pyridoxal phosphate (PL P) cofactors are necessary to catalyze the condensation of homocysteine and serine to generate cystathionine. While the role for the PLP cofactor is t hought to be similar to that in other PLP-dependent enzymes that catalyze b eta -replacement reactions, the exact role for the heme remains unclear. In this study, we have characterized the heme cofactor of CBS in both the fer ric and ferrous states using resonance Raman spectroscopy. Positive identif ication of a cysteine ligand was achieved by global S-34 isotopic substitut ion which allowed us to assign the v(Fe-S) for the six-coordinate low-spin ferric heme at 312 cm(-1). In addition, the CO adduct of ferrous CBS has vi brational frequencies characteristic of a histidine-heme-CO complex in a hy drophobic environment, and indicates that the Fe-S(Cys) bond is labile. We have also found that addition of HgCl2 to the ferric heme causes conversion of the low-spin heme to a five-coordinate high-spin heme with loss of the cysteine ligand. The present spectroscopic studies do not support a reactio n mechanism in which homocysteine binds directly to the heme via displaceme nt of the Cys ligand in the binary enzyme complex, as had been previously p roposed.