| Abstract
| - Human cystathionine β-synthase (CBS) is an essential enzyme for the removal of the toxicmetabolite homocysteine. Heme and pyridoxal phosphate (PLP) cofactors are necessary to catalyze thecondensation of homocysteine and serine to generate cystathionine. While the role for the PLP cofactoris thought to be similar to that in other PLP-dependent enzymes that catalyze β-replacement reactions,the exact role for the heme remains unclear. In this study, we have characterized the heme cofactor ofCBS in both the ferric and ferrous states using resonance Raman spectroscopy. Positive identification ofa cysteine ligand was achieved by global 34S isotopic substitution which allowed us to assign the ν(Fe−S) for the six-coordinate low-spin ferric heme at 312 cm-1. In addition, the CO adduct of ferrous CBShas vibrational frequencies characteristic of a histidine−heme−CO complex in a hydrophobic environment,and indicates that the Fe−S(Cys) bond is labile. We have also found that addition of HgCl2 to the ferricheme causes conversion of the low-spin heme to a five-coordinate high-spin heme with loss of the cysteineligand. The present spectroscopic studies do not support a reaction mechanism in which homocysteinebinds directly to the heme via displacement of the Cys ligand in the binary enzyme complex, as had beenpreviously proposed.
|
