title: Dioxygen reactivity and heme redox potential of truncated human cystathionine beta-synthase.
abstract: Cystathionine beta-synthase (CBS) catalyzes the condensation of serine and homocysteine to cystathionine, which represents the committing step in the transsulfuration pathway. CBS is unique in being a pyridoxal phosphate-dependent enzyme that has a heme cofactor. The activity of CBS under in vitro conditions is responsive to the redox state of the heme, which is distant from the active site and has been postulated to play a regulatory role. The heme in CBS is unusual; it is six-coordinate, low spin, and contains cysteine and histidine as axial ligands. In this study, we have assessed the redox behavior of a human CBS dimeric variant lacking the C-terminal regulatory domain. Potentiometric redox titrations showed a reversible response with a reduction potential of -291 +/- 5 mV versus the normal hydrogen electrode, at pH 7.2. Stopped-flow kinetic determinations demonstrated that Fe(II)CBS reacted with dioxygen yielding Fe(III)CBS without detectable formation of an intermediate species. A linear dependence of the apparent rate constant of Fe(II)CBS decay on dioxygen concentration was observed and yielded a second-order rate constant of (1.11 +/- 0.07) x 10 (5) M (-1) s (-1) at pH 7.4 and 25 degrees C for the direct reaction of Fe(II)CBS with dioxygen. A similar reactivity was observed for full-length CBS. Heme oxidation led to superoxide radical generation, which was detected by the superoxide dismutase (SOD)-inhibitable oxidation of epinephrine. Our results show that CBS may represent a previously unrecognized source of cytosolic superoxide radical.
authors.affiliation: Laboratorio de Enzimología, Facultad de Ciencias, Universidad de la República, Montevideo, Uruguay.
authors.forename: Sebastián
authors.initials: S
authors.lastname: Carballal
authors.forename: Peter
authors.initials: P
authors.lastname: Madzelan
authors.forename: Carlos F
authors.initials: CF
authors.lastname: Zinola
authors.forename: Martín
authors.initials: M
authors.lastname: Graña
authors.forename: Rafael
authors.initials: R
authors.lastname: Radi
authors.forename: Ruma
authors.initials: R
authors.lastname: Banerjee
authors.forename: Beatriz
authors.initials: B
authors.lastname: Alvarez
mesh_terms: D001665:Binding Sites; D003198:Computer Simulation; D003541:Cystathionine beta-Synthase / Q000737:chemistry* / Q000378:metabolism*; D006418:Heme / Q000737:chemistry* / Q000378:metabolism*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008958:Models, Molecular; D010084:Oxidation-Reduction; D011485:Protein Binding
chemical_list: D006418:Heme; D003541:Cystathionine beta-Synthase