Inactivation of cystathionine β-synthase with peroxynitrite

Cystathionine β-synthase (CBS) is a homocysteine metabolizing enzyme that contains pyridoxal phosphate (PLP) and a six-coordinate heme cofactor of unknown function. CBS was inactivated by peroxynitrite, the product of nitric oxide and superoxide radicals. The IC50 was ∼150 μM for 5 μM ferric CBS. Stopped-flow kinetics and competition experiments showed a direct reaction with a second-order rate constant of (2.4-5.0) × 104 M−1 s−1 (pH 7.4, 37 °C). The radicals derived from peroxynitrite, nitrogen dioxide and carbonate radical, also inactivated CBS. Exposure to peroxynitrite did not modify bound PLP but led to nitration of Trp208, Trp43 and Tyr223 and alterations in the heme environment including loss of thiolate coordination, conversion to high-spin and bleaching, with no detectable formation of oxo-ferryl compounds nor promotion of one-electron processes. This study demonstrates the susceptibility of CBS to reactive oxygen/nitrogen species, with potential relevance to hyperhomocysteinemia, a risk factor for cardiovascular diseases.