NO•Binds Human Cystathionine β- Synthase Quickly and Tightly

Background: The H2S-generating human enzyme cystathionine -synthase (CBS) is inhibited by NO center dot and CO. Results: NO center dot binds to the ferrous heme in human CBS much more quickly than CO and much more tightly than currently thought. Conclusion: Results support the physiological role of NO center dot in CBS regulation. Significance: CBS may integrate the cross-talk among NO center dot, CO, and H2S, major modulators in human (patho)physiology. The hexa-coordinate heme in the H2S-generating human enzyme cystathionine -synthase (CBS) acts as a redox-sensitive regulator that impairs CBS activity upon binding of NO center dot or CO at the reduced iron. Despite the proposed physiological relevance of this inhibitory mechanism, unlike CO, NO center dot was reported to bind at the CBS heme with very low affinity (K-d = 30-281 m). This discrepancy was herein reconciled by investigating the NO center dot reactivity of recombinant human CBS by static and stopped-flow UV-visible absorption spectroscopy. We found that NO center dot binds tightly to the ferrous CBS heme, with an apparent K-d 0.23 m. In line with this result, at 25 degrees C, NO center dot binds quickly to CBS (k(on) approximate to 8 x 10(3) m(-1) s(-1)) and dissociates slowly from the enzyme (k(off) approximate to 0.003 s(-1)). The observed rate constants for NO center dot binding were found to be linearly dependent on [NO center dot] up to approximate to 800 m NO center dot, and >100-fold higher than those measured for CO, indicating that the reaction is not limited by the slow dissociation of Cys-52 from the heme iron, as reported for CO. For the first time the heme of human CBS is reported to bind NO center dot quickly and tightly, providing a mechanistic basis for the in vivo regulation of the enzyme by NO center dot. The novel findings reported here shed new light on CBS regulation by NO center dot and its possible (patho)physiological relevance, enforcing the growing evidence for an interplay among the gasotransmitters NO center dot, CO, and H2S in cell signaling.