Kinetics of Reversible Reductive Carbonylation of Heme in Human Cystathionine β‑Synthase

Cystathionine β-synthase (CBS) catalyzes the condensation of homocysteine with serine or cysteine to form cystathionine and water or hydrogen sulfide (H₂S), respectively. In addition to pyridoxal phosphate, human CBS has a heme cofactor with cysteine and histidine as ligands. While Fe­(III)-CBS is inert to exogenous ligands, Fe­(II)-CBS can be reversibly inhibited by carbon monoxide (CO) and reoxidized by O₂ to yield superoxide radical. In this study, we have examined the kinetics of Fe­(II)­CO-CBS formation and reoxidation. Reduction of Fe­(III)-CBS by dithionite showed a square root dependence on concentration, indicating that the reductant species was the sulfur dioxide radical anion (SO₂^•–) that exists in rapid equilibrium with S₂O₄^2–. Formation of Fe­(II)­CO-CBS from Fe­(II)-CBS and 1 mM CO occurred with a rate constant of (3.1 ± 0.4) × 10^–3 s^–1 (pH 7.4, 25 °C). The reaction of Fe­(III)-CBS with the reduced form of the flavoprotein methionine synthase reductase in the presence of CO and NADPH resulted in its reduction and carbonylation to form Fe­(II)­CO-CBS. Fe­(II)-CBS was formed as an intermediate with a rate constant of (9.3 ± 2.5) × 10² M^–1 s^–1. Reoxidation of Fe­(II)­CO-CBS by O₂ was multiphasic. The major phase showed a hyperbolic dependence on O₂ concentration. Although H₂S is a product of the CBS reaction and a potential heme ligand, we did not find evidence of an effect of exogenous H₂S on activity or heme binding. Reversible reduction of CBS by a physiologically relevant oxidoreductase is consistent with a regulatory role for the heme and could constitute a mechanism for cross talk among the CO, H₂S, and superoxide signaling pathways.