Hydrogen sulfide biogenesis by and regulation of cystathionine β-synthase

Human cystathionine β-synthase (CBS) is the first enzyme in the transsulfuration pathway that catalyzes condensation of serine and homocysteine to give cystathionine. CBS is unique pyridoxal-phosphate (PLP) dependent enzyme in that it also contains heme as a second cofactor. The crystal structure of the truncated catalytic core of human CBS revealed that Cys52 and His65 are the heme ligands. The heme in CBS is situated at a 20 Å distance from the PLP site, which excludes a direct role for heme in catalysis and suggests instead a regulatory role. CBS also harbors a tandem repeat of CBS domains, at its C-terminal, which are secondary structural motifs found in diverse families of proteins. S-adenosylmethionine (AdoMet) is a V-type allosteric effector of CBS, which presumably binds to the C-terminal domain. Mutations in CBS are the single most common cause of severe hereditary hyperhomocystenemia and over 130 pathogenic mutations have been described so far. Any perturbation in the heme ligand environment leads to a decrease in or loss of the CBS activity. Based on the crystal structure, we proposed that one pathway for communication between heme and PLP could be via interaction between Arg266 and the heme ligand, Cys52. In this study, we have characterized pathogenic mutations Arg266Lys and Arg266Gly, and third mutant Arg266Met, to test the proposed communication pathway. Our studies show that Arg266, a second coordination sphere ligand to heme, plays a critical role in modulating the electronic and redox properties of heme in CBS. The salt-bridge interaction between Cys52 and Arg266 is necessary for stabilizing the ferrous 449 nm species in wild-type CBS. The Arg266Met mutant is reduced to a species with a Soret maximum at 424 nm, which is inactive and has been referred to as the C-424 species . Using extended X-ray absorption fine structure spectroscopic (EXAFS) analyses, we have assessed the nature of ligands in C-424 species. Another aspect of our study investigates the role of CBS in hydrogen sulfide (H2S) biogenesis. In mammals, two PLP-dependent enzymes are responsible for most of the H2S production: CBS and cystathionine γ-lyase (CSE). We have elucidated the detailed kinetic analysis of the H2S-generating reactions by human and yeast CBS. CBS from both organisms show a marked preference for H2S generation by β-replacement of cysteine by homocysteine. Further, we have used kinetic data to simulate the relative contributions of CBS and CSE to H2S generation. Our results show that under normal conditions assuming equimolar concentrations of CBS and CSE and full activation of CBS by AdoMet, the relative contribution of CBS to CSE is 7:3, whereas under severe hyperhomocysteinemia conditions the relative contribution is 3:7. We also show the existence of an alternative transsulfuration reaction catalyzed by CBS and a new sulfur metabolite, lanthionine produced by CBS, which adds to the sulfur metabolome. We have also studied the pre-steady-state kinetic analysis of the H2 S-generating reactions as well as the canonical β-replacement reaction of serine by homocysteine catalyzed by yCBS. We observe a difference in the rate constants for the binding of serine and cysteine to the enzyme