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

Sebastián Carballal,Ernesto Cuevasanta,Pramod K. Yadav,Carmen Gherasim,David P. Ballou,Beatriz Alvarez,Ruma Banerjee

doi:10.1074/jbc.m116.718734

Abstract

Cystathionine β-synthase (CBS) is a pyridoxal phosphate-dependent enzyme that catalyzes the condensation of homocysteine with serine or with cysteine to form cystathionine and either water or hydrogen sulfide, respectively. Human CBS possesses a noncatalytic heme cofactor with cysteine and histidine as ligands, which in its oxidized state is relatively unreactive. Ferric CBS (Fe(III)-CBS) can be reduced by strong chemical and biochemical reductants to Fe(II)-CBS, which can bind carbon monoxide (CO) or nitric oxide (NO(•)), leading to inactive enzyme. Alternatively, Fe(II)-CBS can be reoxidized by O2to Fe(III)-CBS, forming superoxide radical anion (O2 (̇̄)). In this study, we describe the kinetics of nitrite (NO2 (-)) reduction by Fe(II)-CBS to form Fe(II)NO(•)-CBS. The second order rate constant for the reaction of Fe(II)-CBS with nitrite was obtained at low dithionite concentrations. Reoxidation of Fe(II)NO(•)-CBS by O2showed complex kinetic behavior and led to peroxynitrite (ONOO(-)) formation, which was detected using the fluorescent probe, coumarin boronic acid. Thus, in addition to being a potential source of superoxide radical, CBS constitutes a previously unrecognized source of NO(•)and peroxynitrite.

Highlights

Ferric CBS (Fe(III)-CBS) can be reduced by strong chemical and biochemical reductants to Fe(II)-CBS, which can bind carbon monoxide (CO) or nitric oxide (NO1⁄7), leading to inactive enzyme
Kinetics of Fe(II)-CBS Reaction with 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)NO1⁄7-CBS species, confirmed by the appearance of a 394 nm peak as reported previously [32, 34, 37] (Fig. 1A)
In the presence of reducing equivalents, this is followed by the reaction of Fe(II)-CBS with NO1⁄7 to yield Fe(II)NO1⁄7-CBS, in analogy to the mechanism proposed for globins (44 – 47)

Summary

Experimental Procedures

Materials—All reagents were purchased from Sigma unless otherwise specified. Coumarin boronic acid (CBA) and peroxynitrite were from Cayman Chemical (Ann Arbor, MI). The resulting Fe(II)-CBS solution was mixed with a series of sodium nitrite concentrations at 37 °C, and the UV-visible absorption spectra were recorded outside the anaerobic chamber. An anaerobic solution of Fe(III)-CBS (10 ␮M) in phosphate buffer (0.1 M, pH 7.4, with 0.1 mM (DTPA) was reduced with dithionite (90 ␮M), and heme reduction was monitored spectrophotometrically by a shift in the Soret peak from 428 to 449 nm. Typical reaction mixtures were prepared in the anaerobic chamber and contained Fe(III)-CBS (15 ␮M) in phosphate buffer (0.1 M, pH 7.4, with 0.1 mM DTPA) to which dithionite (100 ␮M) was added. Sodium nitrite (4 mM) was added, and Fe(II)NO1⁄7-CBS formation was confirmed spectrophotometrically, followed by gel filtration to avoid possible interference from nitrite or dithionite. All experiments were repeated at least three times, and representative data are shown

Results

Mammalian hemeproteins

Discussion