Characterization of the heme in human cystathionine beta-synthase by X-rayabsorption and electron paramagnetic resonance spectroscopies

Human cystathionine beta-synthase is one of two key enzymes involved in int racellular metabolism of homocysteine. It catalyzes a beta-replacement reac tion in which the thiolate of homocysteine replaces the hydroxyl group of s erine to give the product, cystathionine. The enzyme is unusual in its depe ndence on two cofactors: pyridoxal phosphate and heme. The requirement for pyridoxal phosphate is expected on the basis of the nature of the condensat ion reaction that is catalyzed; however the function of the heme in this pr otein is unknown. We have examined the spectroscopic properties of the heme in order to assign the axial ligands provided by the protein. The heme Sor et peak of ferric cystathionine beta-synthase is at 428 nm and shifts to si milar to 395 nm upon addition of the thiol chelator, mercuric chloride. Thi s is indicative of B-coordinate low-spin heme converting to a 5-coordinate high-spin heme. The enzyme as isolated exhibits a rhombic EPR signal with g values of 2.5, 2.3, and 1.86, which are similar to those of heme proteins and model complexes with imidazole/thiolate ligands. Mercuric chloride trea tment of the enzyme results in conversion of the rhombic EPR signal to a g 6 signal, consistent with formation of the high-spin ferric heme. The X-ray absorption data reveal that iron in ferric cystathionine B-synthase is 6-c oordinate, with 1 high-Z scatterer and 5 low-Z scatterers. This is consiste nt with the presence of 5 nitrogens and 1 sulfur ligand. Together, these da ta support assignment of the axial ligands as cysteinate and imidazole in f erric cystathionine beta-synthase.