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 square root dependence on concentration, indicating that the reductant species was the sulfur dioxide radical anion (SO2•−) that exists in rapid equilibrium with S2O42−. Formation of Fe(II)CO-CBS from Fe(II)-CBS and 1 mM CO occurred at (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 intermediate with a rate constant of (9.3 ± 2.5) × 102 M−1 s−1. Reoxidation of Fe(II)CO-CBS by O2 was multiphasic. The major phase showed hyperbolic dependence on O2 concentration. Although H2S is a product of the CBS reaction and a potential heme ligand, we did not find evidence for exogenous H2S effect 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 crosstalk between the CO, H2S and superoxide signaling pathways.

S-Glutathionylation Enhances Human Cystathionine β-Synthase Activity Under Oxidative Stress Conditions

“…The N-terminal domain binds the heme cofactor, which functions as an allosteric regulator (44), inhibiting CBS activity in response to binding of CO or NO (3,47) or during reduction of nitrite (14). Nitration of tryptophan…”

NO• Binds Human Cystathionine β-Synthase Quickly and Tightly

“…This heme displays a remarkably low reduction potential (Ϫ350 mV versus standard hydrogen electrode) (13) and acts as a redox-dependent gas sensor because of its ability to react with O 2 and to bind CO and NO ⅐ in the reduced form. Despite the low reduction potential and the fact that most studies on the CBS heme reactivity were carried out in the presence of a large excess of the potent chemical reductant sodium dithionite, it has been recently shown that the diflavin enzyme methionine synthase reductase is able to reduce the CBS heme with NADPH as the electron donor (14,15), conferring greater physiologic relevance to ferrous CBS and its adducts with CO and NO ⅐ .…”

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

“…In addition, the observation that glutathione and cysteine, which are efficient peroxynitrite scavengers, did not inhibit formation of COH fluorescence is also consistent with the in situ formation of peroxynitrite at the heme site. Fe(III)-CBS can be reduced to Fe(II)-CBS by a physiological reducing system (28,29). Thus, in addition to being a source of O 2 .…”

“…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).…”

“…Despite the fairly low heme reduction potential of Ϫ0.350 V (21), CBS reduction can potentially be accomplished in vivo by enzymatic oxidoreductases. We have previously demonstrated that the cytosolic diflavin-containing methionine synthase reductase, in the presence of NADPH as electron donor, is able to reduce the heme in CBS (28,29). In the reduced form, Fe(II)-CBS can react with O 2 , and it can also bind CO and NO ⅐ .…”