Dynamics of Carbon Monoxide Binding to Cystathionine β-Synthase

Abstract

Cystathionine ␤-synthase (CBS) condenses homocysteine, a toxic metabolite, with serine in a pyridoxal phosphate-dependent reaction. It also contains a heme cofactor to which carbon monoxide (CO) or nitric oxide can bind, resulting in enzyme inhibition. To understand the mechanism of this regulation, we have investigated the equilibria and kinetics of CO binding to the highly active catalytic core of CBS, which is dimeric. CBS exhibits strong anticooperativity in CO binding with successive association constants of 0.24 and 0.02 M ؊1 . Stopped flow measurements reveal slow CO association (0.0166 s ؊1 ) limited by dissociation of the endogenous ligand, Cys-52. Rebinding of CO and of Cys-52 following CO photodissociation were independently monitored via time-resolved resonance Raman spectroscopy. The Cys-52 rebinding rate, 4000 s ؊1 , is essentially unchanged between pH 7.6 and 10.5, indicating that the pK a of Cys-52 is shifted below pH 7.6. This effect is attributed to the nearby Arg-266 residue, which is proposed to form a salt bridge with the dissociated Cys-52, thereby inhibiting its protonation and slowing rebinding to the Fe. This salt bridge suggests a pathway for enzyme inactivation upon CO binding, because Arg-266 is located on a helix that connects the heme and pyridoxal phosphate cofactor domains.

NO• Binds Human Cystathionine β-Synthase Quickly and Tightly

“…The kinetic behavior observed after anaerobically mixing ferrous CBS with 1 mM CO in the stopped-flow apparatus was identical to what has been reported in the literature (15,19). In agreement with previous reports (19), CO binding was very slow, causing the expected spectral changes (blueshift of the Soret band from 449 Reactions were investigated at 25°C in 50 mM potassium phosphate, 300 mM KCl, 10% glycerol, 100 M EDTA, pH 7.0, containing 2 mM glucose, 4 units/ml glucose oxidase, 13 g/ml catalase, and SOD (6 units/ml for NO ⅐ and CO assays and 6 -90 units/ml for O 2 assays). A, normalized absorption changes measured at 449 nm after mixing ferrous CBS (1.6 M in heme, before mixing) with NO ⅐ (770, 300, 100, and 50 M before mixing, from left to right).…”

“…In the case of CO, two apparent K d had to be assumed to satisfactorily fit the data, in agreement with Refs. 16,19.…”

“…Despite the several observations pointing to NO ⅐ and CO as physiological regulators of CBS, only the parameters determined for CO binding to the enzyme, particularly its low dissociation constant, appear consistent with such a role (14,19,20). The reported very weak binding of NO ⅐ to ferrous CBS is indeed hardly compatible with the nanomolar to few micromolar physiological concentrations of NO ⅐ .…”

“…The reaction with CO proceeds at very low rates showing hyperbolic dependence on CO concentration (14 -16, 19). CO binding has been proposed to be limited by the slow dissociation rate (0.0166 s Ϫ1 at 24.5°C) of Cys-52 from the CBS heme (19).…”

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

“…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).…”