NO• Binds Human Cystathionine β-Synthase Quickly and Tightly

Abstract

Background:The H 2 S-generating human enzyme cystathionine ␤-synthase (CBS) is inhibited by NO ⅐ and CO. Results: NO ⅐ 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 ⅐ in CBS regulation. Significance: CBS may integrate the cross-talk among NO ⅐ , CO, and H 2 S, major modulators in human (patho)physiology.

Kinetics of Nitrite Reduction and Peroxynitrite Formation by Ferrous Heme in Human Cystathionine β-Synthase

“…1B). The reaction of ferric CBS with NO ⅐ is very slow as reported previously (32) and not expected to be a contributing factor under our experimental conditions. An effect of dithionite on the reaction of Fe(II)-CBS with nitrite was observed.…”

“…Nitrite-When an anaerobic solution of CBS was reduced with dithionite and mixed with nitrite, the characteristic Soret maximum of Fe(II)-CBS at 449 nm was converted to the five-coordinate Fe(II)NO ⅐ -CBS species, confirmed by the appearance of a 394 nm peak as reported previously (32,34,37) (Fig. 1A).…”

“…It is important to note that dithionite does not effectively reduce nitrite (49). Dithionite can compete with Fe(II)-CBS for NO ⅐ because the formation of Fe(II)NO ⅐ -CBS is a relatively slow process (32). Accordingly, when a 10-fold higher value for the k on for the reaction between Fe(II)-CBS and NO ⅐ was used in the simulation, the interference due to dithionite decreased (Fig.…”

“…Fe(II)-CBS can bind CO, which replaces the axial cysteine ligand, forming Fe(II)CO-CBS. The observed rate constants increase hyperbolically with CO concentration, reaching limiting values of 0.012-0.017 s Ϫ1 at 25°C (31,32) or 0.0031 s Ϫ1 at 1 mM CO (28). Relevantly, Fe(II)CO-CBS displays decreased catalytic activity, with a K i for CO of ϳ5.6 M, which might be within a physiologically relevant concentration range (31,33).…”

“…Exposure of Fe(II)CO-CBS to O 2 results in its rapid reoxidation to Fe(III)-CBS concomitant with recovery of enzyme activity (29). In contrast to CO, NO ⅐ binding to Fe(II)-CBS results in a pentacoordinate iron-nitrosyl Fe(II)NO ⅐ -CBS species with a Soret maximum at 394 nm, indicating the loss of both endogenous axial ligands (34 (32). In addition, Fe(II)-CBS can slowly decay to an inactive Fe(II)424-CBS form with a Soret maximum at 424 nm, in which the cysteine is replaced by an unidentified neutral ligand (35,36).…”