Resonance Raman characterization of the heme cofactor in cystathionine beta-synthase. Identification of the Fe-S(Cys) vibration in the six-coordinatelow-spin heme
El. Green et al., Resonance Raman characterization of the heme cofactor in cystathionine beta-synthase. Identification of the Fe-S(Cys) vibration in the six-coordinatelow-spin heme, BIOCHEM, 40(2), 2001, pp. 459-463
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.