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

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.


Introduction
NO regulates a wide range of physiological processes including vasorelaxation, neurotransmission and immune responses [1][2][3]. Its potency as a signaling molecule relies on its short half-life, limited diffusibility and high reactivity with heme proteins [4]. The primary source of NO is nitric oxide synthase (NOS), which oxidizes L-arginine to generate L-citrulline and NO. The discovery of NO-mediated hypoxic vasorelaxation suggested the presence of additional sources of NO under oxygen-limiting conditions where NOS is inactive [5]. Biochemical and physiological evidence exists suggesting that nitrite, the one electron oxidation product of NO, represents a circulating pool of NO that can be accessed under hypoxic conditions [6]. In addition to nonenzymatic acidic reduction of nitrite, enzyme-mediated nitrite reduction has also been reported [7,8]. To date, a limited number of hemeproteins, such as globins and cytochrome c have been identified as nitrite reductases albeit their activities cannot entirely account for the positive effects of nitrite treatments [5,[9][10][11][12]. Furthermore, clinical studies that have investigated the usefulness of nitrite as an NO donor identified a non-linear dose-dependent increase of nitrite concentration with administered nitrite suggesting additional players for nitrite clearance [13,14].
Human CBS is a 59-pyridoxal phosphate (PLP)-and hemecontaining protein that controls the levels of key sulfur metabolites including homocysteine, glutathione and H 2 S [15][16][17]. Genetic defects in CBS represent the most common cause of hereditary homocystinuria, an inborn error of metabolism associated with aggressive occlusive arterial disease [18]. CBS uses its PLP cofactor to catalyze ß-replacement reactions that contribute to homocysteine clearance in the presence of either serine or cysteine as a co-substrate. The ß-replacement of serine with homocysteine represents the cannonical reaction in the transsulfuration pathway, while the ß-replacement of cysteine with homocysteine results in H 2 S biogenesis [19,20]. A unique heme b cofactor in human CBS represents a puzzling evolutionarily accessory whose role is unclear [21]. While the heme is not required for enzyme activity, both structural and regulatory roles have been proposed for it [22][23][24]. Binding of carbon monoxide (CO) or NO to the Fe II -CBS heme inhibits enzyme activity [25,26]. We have demonstrated that despite the low reduction potential (2350 mV) for the Fe 3+ /Fe 2+ couple of CBS [27], reversible regulation by CO binding can be achieved with physiologically relevant reductants like methionine synthase reductase (MSR) and novel reductase 1 [24,28].
The growing interest in H 2 S signaling, which mediates profound physiological effects [29,30] has focused attention on the enzymes responsible for its biogenesis and decay [31][32][33]. In addition to CBS, the enzymes involved in H 2 S production include cystathionase [34,35] and mercaptopyruvate sulfurtransferase [36,37]. Rapid turnover of H 2 S contributes to maintaining its low steady-state concentrations estimated to be in the ,10-30 nM range [38,39]. Hence, regulation of both H 2 S production and catabolism are important targets for cellular and pharmacological modulation of its levels [40]. Increasingly, there is evidence for interactions between the H 2 S and the gas-signaling pathways elicited by CO and NO but the molecular mechanisms for this cross-talk are poorly understood [41].
Changes in the heme ligation or spin state are conveyed over a long distance to the active site of CBS and inhibits its PLPdependent activity [26,42]. The ability of flavin oxidoreductases to generate Fe II -CBS in the presence of NADPH [24] suggested that the allosteric heme sensor domain might exhibit additional regulatory strategies. Herein, we report a previously unknown function of the heme in human CBS, i.e. reaction with nitrite to form Fe II -NO, which inhibits H 2 S formation. Our results suggest a possible molecular mechanism for crossover between the NO and H 2 S signaling pathways.

Nitrite Reductase Activity of CBS
The release of NO from nitrite is mediated by interactions between Fe II -hemoglobins and nitrite [5,9,10] and the possible involvement of other heme proteins in this process is implicated [11]. Long-term sodium nitrite administration displayed a nonlinear increase in nitrite concentrations, which was modeled as an increase in its clearance [14]. We therefore examined the interaction of Fe II -CBS with nitrite. Reaction of 10 mM Fe II -CBS with 10 mM nitrite under anaerobic conditions, showed time-and nitrite concentration-dependent changes in the heme spectrum consistent with formation of Fe II -NO CBS (Fig.1A). The decrease in absorbance of Fe II -CBS with a Soret maximum at 450 nm and a and ß bands at 540 and 570 nm, respectively was accompanied by the appearance of a 394 nm peak and broadening of the a/b bands, corresponding to formation of five-coordinate Fe II -NO CBS. The data for Fe II -CBS disappearance and Fe II -NO CBS formation were fitted to single exponential functions, and yielded k obs = 0.52 min 21 (Fig. 1B). In analogy to globins [46][47][48], the mechanism for nitrite reduction by CBS is summarized by equations 1 and 2.
The kinetics of the nitrite reduction reaction in the presence of excess sodium dithionite is consistent with one Fe II -CBS forming one Fe II -NO CBS (Fig. 1B). Furthermore, the isosbestic conversion of the ferrous species to Fe II -NO indicates that Fe III -CBS does not accumulate, i.e. it is rapidly reduced to Fe II -CBS, which reacts with NO. The bimolecular rate constant calculated from the linear fit of k obs as a function of nitrite concentration is 0.6 M 21 s 21 at 37uC, pH 7.4 (Fig. 1A, inset). In the presence of the allosteric activator of CBS, S-adenosylmethionine, the nitrite reductase activity was increased ,2-fold (k obs = 0.98 min 21 ).
Since the Fe II -CBS-dependent nitrite reduction is predicted to require a proton (equation 1), its pH-dependence was studied. A 10-fold increase in the rate of the reaction was observed between pH 8 and 7 (Fig. 1C). The slope of the rate dependence on proton concentration was 1.260.03 consistent with the requirement for one proton per Fe II -NO CBS formation. The nitrite reductase activity of CBS in vivo would be contingent upon the presence of a reducing system that generates the reactive Fe II species. MSR can shuttle an electron from NADPH through its flavin cofactors to Fe III -CBS [24] (Fig. 2A). In the presence of NADPH, nitrite and substoichiometric MSR, the conversion of Fe III -to Fe II -NO CBS was observed indicating that nitrite reduction can be achieved in the presence of a biochemical reducing system (Figs. 2B,C). We note that the UV-visible spectrum of the Fe II -NO-CBS in this reaction mixture is partially obscured by NADPH oxidation during this experiment. EPR spectroscopy provides further evidence for the formation of the Fe II -NO-CBS product as discussed below (Fig. 2B). The observed rate for NADPH/MSR-dependent Fe II -NO CBS formation as monitored by the decrease in absorbance at 429 nm is 0.007 min 21 (Fig. 2D). The latter is slower than the rate obtained in the presence of dithionite, a more efficient artificial reductant for CBS whose kinetic characterization has been reported by Carballal et al. The results suggest a shift in the rate-limiting step from nitrite reduction in the presence of dithionite to the reduction of CBS-Fe III -to Fe II by NADPH/MSR.

EPR Spectrum of CBS during Nitrite Reduction
The heme in human CBS is six-coordinate in both the Fe II and Fe III states, and Cys52 and His65 serve as axial ligands [22,[49][50][51] ( Fig. 2A). The EPR spectrum obtained during CBS-catalyzed nitrite reduction in the presence of dithionite or NADPH/MSR, provides evidence for the formation of paramagnetic, fivecoordinate Fe II -NO CBS with a characteristic three-line hyperfine splitting resulting from the interaction between the unpaired electron and the I = 1 nucleus of the nitrogen in NO (Fig. 2B). Formation of Fe II -NO CBS leads to loss of both endogenous ligands [26], unlike other six-coordinate hemeproteins where NO binding displaces only one of the endogenous axial ligands. Nitrite Reductase Activity at the Heme Site of CBS Inhibits b-replacement Activity and H 2 S Biogenesis at the PLP Site Binding of NO to Fe II -CBS inhibits its activity in the canonical reaction that generates cystathionine from homocysteine and serine [26]. The K D for NO binding to CBS (281650 mM) [26] was previously determined at pH 8.6, the pH maximum for CBS activity. However, the presence of excess dithionite (1.5 mM) in the reaction mixture could have resulted in slow reduction of NO as reported previously [52], leading to an overestimation of the K D value for NO binding to CBS. We therefore reassessed binding of NO to CBS at physiological pH (7.4) and employed NADPH and MSR as a source of electrons (Fig. 3A). In the presence of the NO precursor, diethylamine NONOate, a shift in the Soret maximum from 428 nm (corresponding to ferric CBS) to 395 nm (Fe II -NO CBS) was observed, consistent with the conversion of sixcoordinate low-spin Fe III to five-coordinate high-spin Fe II -NO CBS as seen previously [26]. Based on this analysis, a K Dapp for binding of NO to CBS was estimated to be 3065 mM. We note that this is an apparent K D and represents an upper limit, since formation of Fe II -NO CBS under these conditions involves multiple equilibria including NADPH binding to MSR, MSR binding to CBS, reduction of CBS by MSR and NO binding to Fe II -CBS.
Formation of Fe II -NO inhibits CBS activity in the canonical serine+homocysteine reaction (82620 mmol mg 21 h 21 for Fe II -NO CBS versus 257625 mmol mg 21 h 21 for Fe III -CBS). The reversibility of inhibition by the Fe II -NO species was assessed by air-oxidation, which led to the ready formation of Fe III -CBS (Fig. 3B). The latter in turn, was accompanied by recovery of activity (195610 mmol mg 21 h 21 ). The incomplete overall recovery of Fe III -CBS activity from Fe II -NO CBS might be due to nitrite-induced degradation of the heme in air as also reported for human hemoglogin [53]. Partial loss of the heme during the reoxidation process has been also observed with Fe II -CO CBS [24]. The mechanism and physiological role of nitrite-induced heme degradation in air for CBS is presently unclear. While the rate of oxidation of Fe II -NO CBS has not been reported yet, oxidation of Fe II -CBS occurs rapidly with a second-order rate constant of 1.1610 5 M 21 s 21 (at 25uC and pH 7.4). Assuming similar oxidation kinetics for Fe II -NO CBS, we propose that nitrite reduction by CBS can modulate its activity via reversible formation of Fe II -NO heme. Displacement of the NO ligand by CO was observed upon incubating Fe II -NO CBS with CO, indicating integrity of the heme in the Fe II -state (Fig. 4A). In addition to the production of cystathionine in the canonical reaction, CBS generates H 2 S using alternative substrates such as cysteine or cysteine+homocysteine. Formation of Fe II -NO CBS was correlated with ,90% inhibition of H 2 S production in the presence of cysteine+homocysteine (Fig. 4B).

Discussion
In this study, we have demonstrated that human CBS reacts with nitrite to generate Fe II -NO at rates that are higher than those reported for the hemoglobin T state (k = 0.082 M 21 s 21 ) and for neuroglobin (k = 0.12 M 21 s 21 and 0.062 M 21 s 21 for the oxidized and reduced protein). The nitrite concentrations used in these experiments to demonstrate the nitrite reductase activity of human CBS are high, albeit similar to those used previously to measure NO-generation from nitrite by other heme-containing proteins [10,11]. We note that high K M values (in the millimolar range) for the CBS substrates (e.g. homocysteine) have also been reported consistently by different groups despite the low (micro-molar) substrate concentrations present inside cells [54]. Since defects in CBS clearly affect cellular utilization of homocysteine and lead to homocysinuria [18], it raises the possibility that either small molecule or protein modulators in the cell increase the affinity of CBS for its substrates for CBS. However, it is too early to speculate on whether cellular modulation of the affinity of CBS for nitrite occurs.
The in vitro nitrite reductase activity of CBS raises the possibility that it might contribute to NO biogenesis from the nitrite pool particularly under hypoxic conditions, and suggests a possible role for CBS in NO-signaling particularly in tissues where CBS is abundant. The high affinity of hemoglobins for NO begs the question as to how NO can be released efficiently to act as a signaling molecule. In this context, the increase in the dissociation rate constant of NO from Fe III versus Fe II -hemoglobin has been proposed as a possible solution, which requires partially oxygenated conditions [55]. The k off for NO from the Fe II -CBS complex is not known. A rapid rate of dissociation would be advantageous by permitting more facile release of NO from CBS under hypoxic conditions.
A potential role of CBS in NO signaling is supported by the ability of a physiological reducing system to mediate formation of Fe II -NO CBS (Fig. 2). Transient formation of Fe II -NO CBS could serve as an allosteric switch for CBS. The contribution of CBS to H 2 S is tissue-dependent [56]. Inhibition of CBS under hypoxic conditions when sulfide oxidation is limited, could represent a mechanism for simultaneously decreasing H 2 S production by CBS. The metabolic consequences of CBS inhibition are likely to be complex and would depend on the relative distribution of CBS versus the other H 2 S producing enzymes. Thus, Fe II -NO CBS would result in increased homocysteine but decreased cysteine, cystathionine and CBS-derived H 2 S (Fig. 4C). Cystathionine, a product of the canonical serine+homocysteine or the noncanonical cysteine+homocysteine reactions, is cleaved by cystathionase to cysteine. Hence CBS inhibition is predicted to decrease cysteine and H 2 S production by CBS and by the cysteine catabolic pathway, comprising cysteine amino transferase and mercaptopyruvate sulfurtransferase. On the other hand, accumulation of homocysteine would increase the rate of cystathionase-dependent H 2 S formation. However, in tissues in which cystathionase levels are low, e.g. brain [57], formation of Fe II -NO CBS is expected to result in homocysteine accumulation and a net decrease in H 2 S synthesis. Similar metabolic changes are predicted for Fe II -CO CBS formation.
Both NO and H 2 S are positive effectors of the cardiovascular system and their specific targets are soluble guanylate cyclase and potassium channels, respectively [58]. Our data suggest a molecular mechanism by which CBS might be important for controlling the balance between the NO and H 2 S signaling pathways. Generation of H 2 S and possibly, NO by CBS also suggests a potential role for the enzyme in regulating production of HSNO, a nitrosothiol described as a signaling molecule [59]. Finally, CBS inhibition diminishes intracellular cysteine concentrations in various cell types [60,61]. Cysteine in turn, is a substrate for H 2 S-generation by cystathionase, a major H 2 S producer, and a limiting substrate for glutathione synthesis (Fig. 4C). Consequently, CBS has the potential to regulate NO, H 2 S and glutathione production either directly or indirectly via its heme-dependent catalytic activity.

Materials
All reagents were purchased from Sigma unless otherwise specified. Diethylamine NONOate was from Cayman Chemical (Ann Arbor, MI). CBS and MSR were purified as previously described [43,44].

UV-visible Spectroscopic Characterization of Nitrite Reduction by Fe II -CBS
Sodium dithionite and nitrite stock solutions were prepared under anaerobic conditions. The reaction mixtures containing anaerobic solution of 10 mM CBS and 3 mM dithionite in 0.1 M HEPES, pH 7.4 were incubated in a 500 ml cuvette. Following reduction of Fe III -CBS to Fe II -CBS, the reactions were initiated by addition of nitrite and its reduction was monitored spectrophotometrically at 37uC. For nitrite-dependence studies, the following nitrite concentrations were used 1, 2.5, 5, 10 and 25 mM. The pH-dependent studies were performed between pH 7.0-8.0, using 0.1 M HEPES buffer at pH 7.0, 7.25, 7.4, 7.75 and 8.0. The low redox potential of the heme iron in CBS makes its reduction by dithionite below pH 7.0 difficult, limiting the pH range for these experiments. For the reduction of nitrite by Fe II -CBS generated by the MSR/NADPH reducing system, the reaction mixtures contained 10-15 mM CBS, 5 mM MSR and 1 mM NADPH.

EPR Spectroscopy of Fe II -NO CBS
Samples containing 15-20 mM CBS in 0.1 M HEPES pH 7.4 containing either 3 mM dithionite and 10 mM nitrite or 10 mM MSR and 1 mM NADPH were incubated at 37uC and the formation of Fe II -NO CBS product was monitored before transferring the reaction mixtures to sealed EPR tubes. The EPR spectra were recorded on a Bruker ESP 300E spectrometer equipped with an Oxford ITC4 temperature controller. The conditions used for spectral acquisition are described in the figure legend.

NO Binding to Fe II -CBS
Binding of NO to 10 mM CBS in 0.1 M HEPES pH 7.4 was determined using NADPH (200 mM) and MSR (5 mM) as a source of electrons and the NO precursor, diethylamine NONOate (diethylammonium (Z)-1-(N,N-diethyl-NONOate amino) diazen-1-ium-1,2-diolate)) (0-533 mM). Reaction mixtures were prepared in a gas-tight syringe and the NO donor was added last. The syringes were sealed and kept without a headspace to prevent NO escape and the Fe II -NO CBS formed was monitored spectroscopically until no further changes were observed.

Activity Tests on Fe III CBS and Fe II -NO CBS Species
The activities of ferric-and ferrous-NO CBS were measured under anaerobic conditions using the radiolabeled assay (using [ 14 C]-serine+homocysteine) and the lead acetate assay (using cysteine+homocysteine) as previously described [43,45].