title: Resonance Raman characterization of the heme cofactor in cystathionine beta-synthase. Identification of the Fe-S(Cys) vibration in the six-coordinate low-spin heme.
abstract: Human cystathionine beta-synthase (CBS) is an essential enzyme for the removal of the toxic metabolite homocysteine. Heme and pyridoxal phosphate (PLP) cofactors are necessary to catalyze the condensation of homocysteine and serine to generate cystathionine. While the role for the PLP cofactor is thought to be similar to that in other PLP-dependent enzymes that catalyze beta-replacement reactions, the exact role for the heme remains unclear. In this study, we have characterized the heme cofactor of CBS in both the ferric and ferrous states using resonance Raman spectroscopy. Positive identification of a cysteine ligand was achieved by global (34)S isotopic substitution which allowed us to assign the nu(Fe-S) for the six-coordinate low-spin ferric heme at 312 cm(-1). In addition, the CO adduct of ferrous CBS has 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 HgCl(2) 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 reaction mechanism in which homocysteine binds directly to the heme via displacement of the Cys ligand in the binary enzyme complex, as had been previously proposed.
authors.affiliation: Department of Biochemistry and Molecular Biology, Oregon Graduate Institute of Science and Technology, Beaverton, Oregon 97006-8921, USA.
authors.forename: E L
authors.initials: EL
authors.lastname: Green
authors.forename: S
authors.initials: S
authors.lastname: Taoka
authors.forename: R
authors.initials: R
authors.lastname: Banerjee
authors.forename: T M
authors.initials: TM
authors.lastname: Loehr
mesh_terms: D001667:Binding, Competitive; D002248:Carbon Monoxide / Q000737:chemistry; D003541:Cystathionine beta-Synthase / Q000737:chemistry*; D003545:Cysteine / Q000737:chemistry*; D005290:Ferric Compounds / Q000737:chemistry; D005296:Ferrous Compounds / Q000737:chemistry; D006418:Heme / Q000737:chemistry*; D006710:Homocysteine / Q000737:chemistry; D006801:Humans; D007506:Iron-Sulfur Proteins / Q000737:chemistry*; D008024:Ligands; D008627:Mercuric Chloride / Q000737:chemistry; D008982:Molybdenum / Q000737:chemistry; D010084:Oxidation-Reduction; D011485:Protein Binding; D011732:Pyridoxal Phosphate / Q000737:chemistry; D013059:Spectrum Analysis, Raman
chemical_list: D005290:Ferric Compounds; D005296:Ferrous Compounds; D007506:Iron-Sulfur Proteins; D008024:Ligands; D006710:Homocysteine; D006418:Heme; D008627:Mercuric Chloride; D011732:Pyridoxal Phosphate; D002248:Carbon Monoxide; D008982:Molybdenum; D003541:Cystathionine beta-Synthase; D003545:Cysteine