Nitrite Reductase Activity and Inhibition of H2S Biogenesis by Human Cystathionine ß-Synthase

Abstract

Nitrite was recognized as a potent vasodilator >130 years and has more recently emerged as an endogenous signaling molecule and modulator of gene expression. Understanding the molecular mechanisms that regulate nitrite metabolism is essential for its use as a potential diagnostic marker as well as therapeutic agent for cardiovascular diseases. In this study, we have identified human cystathionine ß-synthase (CBS) as a new player in nitrite reduction with implications for the nitrite-dependent control of H2S production. This novel activity of CBS exploits the catalytic property of its unusual heme cofactor to reduce nitrite and generate NO. Evidence for the possible physiological relevance of this reaction is provided by the formation of ferrous-nitrosyl (FeII-NO) CBS in the presence of NADPH, the human diflavin methionine synthase reductase (MSR) and nitrite. Formation of FeII-NO CBS via its nitrite reductase activity inhibits CBS, providing an avenue for regulating biogenesis of H2S and cysteine, the limiting reagent for synthesis of glutathione, a major antioxidant. Our results also suggest a possible role for CBS in intracellular NO biogenesis particularly under hypoxic conditions. The participation of a regulatory heme cofactor in CBS in nitrite reduction is unexpected and expands the repertoire of proteins that can liberate NO from the intracellular nitrite pool. Our results reveal a potential molecular mechanism for cross-talk between nitrite, NO and H2S biology.

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

“…This value for the second order rate constant between nitrite and Fe(II)-CBS is an approximation because of the multiple reactions required to obtain Fe(II)NO ⅐ -CBS from Fe(II)-CBS and nitrite. In the presence of the allosteric activator AdoMet, the second order rate constant obtained from fits at 449 nm is 1.32 Ϯ 0.06 M Ϫ1 s Ϫ1 , ϳ2-fold greater than in its absence and in agreement with a previous report (37). Interestingly, the T257I mutant of CBS exhibited a Ͼ10-fold increase in the value of the second order rate constant than wild-type CBS in the absence of AdoMet (Table 2).…”

“…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)NO ⅐ -CBS species, confirmed by the appearance of a 394 nm peak as reported previously (32,34,37) (Fig. 1A).…”

“…Although Fe(II)424-CBS formation is promoted under nonphysiological conditions such as high temperature (36), the biological significance of this process is not clear. We have demonstrated recently that Fe(II)-CBS has the ability to reduce nitrite (NO 2 Ϫ ) to NO ⅐ , leading to Fe(II)NO ⅐ -CBS formation, suggesting a possible new role for CBS in NO ⅐ signaling (37).…”

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…”

Pharmacology and therapeutic role of inorganic nitrite and nitrate in vasodilatation

“…The most recent member of the heme-based nitrite reductases is cystathionine beta-synthase (CBS) (Gherasim et al, 2014), a key enzyme of the transsulfuration pathway involved in homocysteine metabolism, glutathione production, and formation of hydrogen sulfide (H 2 S). As with other heme-based reductases, NO-heme formation leads to autoinhibition of the enzyme, representing an interesting new facet of the NO/H 2 S cross-talk.…”