title: Kinetics of reversible reductive carbonylation of heme in human cystathionine β-synthase.
abstract: Cystathionine β-synthase (CBS) catalyzes the condensation of homocysteine with serine or cysteine to form cystathionine and water or hydrogen sulfide (H2S), respectively. In addition to pyridoxal phosphate, human CBS has a heme cofactor with cysteine and histidine as ligands. While Fe(III)-CBS is inert to exogenous ligands, Fe(II)-CBS can be reversibly inhibited by carbon monoxide (CO) and reoxidized by O2 to yield superoxide radical. In this study, we have examined the kinetics of Fe(II)CO-CBS formation and reoxidation. Reduction of Fe(III)-CBS by dithionite showed a square root dependence on concentration, indicating that the reductant species was the sulfur dioxide radical anion (SO2(•-)) that exists in rapid equilibrium with S2O4(2-). Formation of Fe(II)CO-CBS from Fe(II)-CBS and 1 mM CO occurred with a rate constant of (3.1 ± 0.4) × 10(-3) s(-1) (pH 7.4, 25 °C). The reaction of Fe(III)-CBS with the reduced form of the flavoprotein methionine synthase reductase in the presence of CO and NADPH resulted in its reduction and carbonylation to form Fe(II)CO-CBS. Fe(II)-CBS was formed as an intermediate with a rate constant of (9.3 ± 2.5) × 10(2) M(-1) s(-1). Reoxidation of Fe(II)CO-CBS by O2 was multiphasic. The major phase showed a hyperbolic dependence on O2 concentration. Although H2S is a product of the CBS reaction and a potential heme ligand, we did not find evidence of an effect of exogenous H2S on activity or heme binding. Reversible reduction of CBS by a physiologically relevant oxidoreductase is consistent with a regulatory role for the heme and could constitute a mechanism for cross talk among the CO, H2S, and superoxide signaling pathways.
authors.affiliation: Laboratorio de Enzimología, Facultad de Ciencias, Facultad de Medicina, Universidad de la República, Montevideo, Uruguay.
authors.forename: Sebastián
authors.initials: S
authors.lastname: Carballal
authors.forename: Ernesto
authors.initials: E
authors.lastname: Cuevasanta
authors.forename: Inés
authors.initials: I
authors.lastname: Marmisolle
authors.forename: Omer
authors.initials: O
authors.lastname: Kabil
authors.forename: Carmen
authors.initials: C
authors.lastname: Gherasim
authors.forename: David P
authors.initials: DP
authors.lastname: Ballou
authors.forename: Ruma
authors.initials: R
authors.lastname: Banerjee
authors.forename: Beatriz
authors.initials: B
authors.lastname: Alvarez
mesh_terms: D002248:Carbon Monoxide / Q000737:chemistry* / Q000378:metabolism; D003541:Cystathionine beta-Synthase / Q000737:chemistry* / Q000378:metabolism; D003545:Cysteine / Q000378:metabolism; D006418:Heme / Q000737:chemistry* / Q000378:metabolism; D006639:Histidine / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008024:Ligands; D010100:Oxygen / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding; D050050:Protein Carbonylation; D015398:Signal Transduction; D013059:Spectrum Analysis, Raman; D013458:Sulfur Dioxide / Q000737:chemistry / Q000378:metabolism; D013481:Superoxides / Q000737:chemistry
chemical_list: D008024:Ligands; D013458:Sulfur Dioxide; D013481:Superoxides; D006418:Heme; D006639:Histidine; D002248:Carbon Monoxide; D003541:Cystathionine beta-Synthase; D003545:Cysteine; D010100:Oxygen