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Hydrogen sulfide bypasses the rate-limiting oxygen activation of heme oxygenase

Open AccessPublished:September 20, 2018DOI:https://doi.org/10.1074/jbc.RA118.004641
      Discovery of unidentified protein functions is of biological importance because it often provides new paradigms for many research areas. Mammalian heme oxygenase (HO) enzyme catalyzes the O2-dependent degradation of heme into carbon monoxide (CO), iron, and biliverdin through numerous reaction intermediates. Here, we report that H2S, a gaseous signaling molecule, is part of a novel reaction pathway that drastically alters HO's products, reaction mechanism, and catalytic properties. Our prediction of this interplay is based on the unique reactivity of H2S with one of the HO intermediates. We found that in the presence of H2S, HO produces new linear tetrapyrroles, which we identified as isomers of sulfur-containing biliverdin (SBV), and that only H2S, but not GSH, cysteine, and polysulfides, induces SBV formation. As BV is converted to bilirubin (BR), SBV is enzymatically reduced to sulfur-containing bilirubin (SBR), which shares similar properties such as antioxidative effects with normal BR. SBR was detected in culture media of mouse macrophages, confirming the existence of this H2S-induced reaction in mammalian cells. H2S reacted specifically with a ferric verdoheme intermediate of HO, and verdoheme cleavage proceeded through an O2-independent hydrolysis-like mechanism. This change in activation mode diminished O2 dependence of the overall HO activity, circumventing the rate-limiting O2 activation of HO. We propose that H2S could largely affect O2 sensing by mammalian HO, which is supposed to relay hypoxic signals by decreasing CO output to regulate cellular functions. Moreover, the novel H2S-induced reaction identified here helps sustain HO's heme-degrading and antioxidant-generating capacity under highly hypoxic conditions.

      Introduction

      Hydrogen sulfide (H2S) has emerged as the third gaseous mediator molecule alongside nitric oxide (NO) and carbon monoxide (CO) in mammalian signaling to modulate a variety of biological processes (
      • Paul B.D.
      • Snyder S.H.
      H2S signalling through protein sulfhydration and beyond.
      ,
      • Kimura H.
      Hydrogen sulfide: its production, release and functions.
      ,
      • Kajimura M.
      • Fukuda R.
      • Bateman R.M.
      • Yamamoto T.
      • Suematsu M.
      Interactions of multiple gas-transducing systems: hallmarks and uncertainties of CO, NO, and H2S gas biology.
      ,
      • Suematsu M.
      • Goda N.
      • Sano T.
      • Kashiwagi S.
      • Egawa T.
      • Shinoda Y.
      • Ishimura Y.
      Carbon monoxide: an endogenous modulator of sinusoidal tone in the perfused rat liver.
      ,
      • Kabe Y.
      • Nakane T.
      • Koike I.
      • Yamamoto T.
      • Sugiura Y.
      • Harada E.
      • Sugase K.
      • Shimamura T.
      • Ohmura M.
      • Muraoka K.
      • Yamamoto A.
      • Uchida T.
      • Iwata S.
      • Yamaguchi Y.
      • Krayukhina E.
      • et al.
      Haem-dependent dimerization of PGRMC1/Sigma-2 receptor facilitates cancer proliferation and chemoresistance.
      ,
      • Filipovic M.R.
      • Zivanovic J.
      • Alvarez B.
      • Banerjee R.
      Chemical biology of H2S signaling through persulfidation.
      ). H2S is endogenously synthesized by cystathionine β-synthase (CBS),
      The abbreviations used are: CBS
      cystathionine β-synthase
      BV
      biliverdin
      BR
      bilirubin
      SBV
      sulfur-containing biliverdin
      SBR
      sulfur-containing bilirubin
      HO
      heme oxygenase
      CPR
      cytochrome P450 reductase
      BVR
      biliverdin reductase
      AAPH
      2,2′-azobis(2-amidinopropane) dihydrochloride
      ESI
      electrospray ionization.
      cystathionine γ-lyase, and 3-mercaptopyruvate sulfurtransferase. Among the gaseous mediators, only H2S can be ionized in aqueous solution to exist mainly as its monoanion form (SH) with negligible amounts of S2− present under physiological pH (pKa values for H2S and SH are 7.0 and >17, respectively) (
      • Filipovic M.R.
      • Zivanovic J.
      • Alvarez B.
      • Banerjee R.
      Chemical biology of H2S signaling through persulfidation.
      ). The SH anion is known to possess high reducing ability and nucleophilicity. One of the most established targets of SH is a cysteine residue of proteins to form persulfide (–SSH). Functions of target proteins such as ATP-dependent potassium (KATP) channels (
      • Paul B.D.
      • Snyder S.H.
      H2S signalling through protein sulfhydration and beyond.
      ) are modulated by the covalent modification, which may proceed through nucleophilic attack of SH to oxidized forms of the cysteine residue, although the detailed mechanism has yet to be identified (
      • Mishanina T.V.
      • Libiad M.
      • Banerjee R.
      Biogenesis of reactive sulfur species for signaling by hydrogen sulfide oxidation pathways.
      ). In addition, SH is reported to react with a variety of small biomolecules to afford reactive sulfur species, including polysulfides (Sn2−) (
      • Filipovic M.R.
      • Zivanovic J.
      • Alvarez B.
      • Banerjee R.
      Chemical biology of H2S signaling through persulfidation.
      ,
      • Nishida M.
      • Sawa T.
      • Kitajima N.
      • Ono K.
      • Inoue H.
      • Ihara H.
      • Motohashi H.
      • Yamamoto M.
      • Suematsu M.
      • Kurose H.
      • van der Vliet A.
      • Freeman B.A.
      • Shibata T.
      • Uchida K.
      • Kumagai Y.
      • et al.
      Hydrogen sulfide anion regulates redox signaling via electrophile sulfhydration.
      ,
      • Shiota M.
      • Naya M.
      • Yamamoto T.
      • Hishiki T.
      • Tani T.
      • Takahashi H.
      • Kubo A.
      • Koike D.
      • Itoh M.
      • Ohmura M.
      • Kabe Y.
      • Sugiura Y.
      • Hiraoka N.
      • Morikawa T.
      • Takubo K.
      • et al.
      Gold-nanofève surface-enhanced Raman spectroscopy visualizes hypotaurine as a robust anti-oxidant consumed in cancer survival.
      ). Considering these unique reactivities, H2S could have further signaling targets, whose identification is crucial for the H2S biology. We have envisaged a new interplay of H2S within the gaseous mediator systems because of their tight interactions, especially in vasorelaxation and oxygen (O2) sensing. For instance, CO binding to a prosthetic heme of CBS inhibits the H2S synthesis to cause vasoconstriction in neurovascular units (
      • Morikawa T.
      • Kajimura M.
      • Nakamura T.
      • Hishiki T.
      • Nakanishi T.
      • Yukutake Y.
      • Nagahata Y.
      • Ishikawa M.
      • Hattori K.
      • Takenouchi T.
      • Takahashi T.
      • Ishii I.
      • Matsubara K.
      • Kabe Y.
      • Uchiyama S.
      • et al.
      Hypoxic regulation of the cerebral microcirculation is mediated by a carbon monoxide-sensitive hydrogen sulfide pathway.
      ). The biological source of CO is heme degradation catalyzed by heme oxygenase (HO), which exists as two isoforms, inducible HO-1 and constitutive HO-2 (
      • Maines M.D.
      The heme oxygenase system: a regulator of second messenger gases.
      ). HO activity could have O2 dependence that is a prerequisite to function as a primary O2 sensor in the cerebral blood vessels as well as in the carotid body (
      • Morikawa T.
      • Kajimura M.
      • Nakamura T.
      • Hishiki T.
      • Nakanishi T.
      • Yukutake Y.
      • Nagahata Y.
      • Ishikawa M.
      • Hattori K.
      • Takenouchi T.
      • Takahashi T.
      • Ishii I.
      • Matsubara K.
      • Kabe Y.
      • Uchiyama S.
      • et al.
      Hypoxic regulation of the cerebral microcirculation is mediated by a carbon monoxide-sensitive hydrogen sulfide pathway.
      ,
      • Williams S.E.
      • Wootton P.
      • Mason H.S.
      • Bould J.
      • Iles D.E.
      • Riccardi D.
      • Peers C.
      • Kemp P.J.
      Hemoxygenase-2 is an oxygen sensor for a calcium-sensitive potassium channel.
      ). These intricate interplays may be an inherent feature of the gaseous mediators and suggest the existence of further unidentified cross-talk. Here, we report that H2S opens a novel pathway of HO to produce a new heme catabolite.
      The new HO reaction, termed an S-HO reaction hereafter, is predicted on the basis of mechanistic knowledge of HO catalysis as well as H2S reactivity. HO catalyzes degradation of heme into biliverdin (BV), ferrous iron, and CO by three successive monooxygenation steps via hydroxyheme and verdoheme intermediates (see Fig. 1A) (
      • Matsui T.
      • Unno M.
      • Ikeda-Saito M.
      Heme oxygenase reveals its strategy for catalyzing three successive oxygenation reactions.
      ,
      • Montellano P.R.
      The mechanism of heme oxygenase.
      ). These O2 activation steps are performed by the substrate heme itself, so that HO catalysis actually involves numerous reaction intermediates (Fig. S1) (
      • Matsui T.
      • Unno M.
      • Ikeda-Saito M.
      Heme oxygenase reveals its strategy for catalyzing three successive oxygenation reactions.
      ,
      • Montellano P.R.
      The mechanism of heme oxygenase.
      ). Among them, we have initially postulated the verdoheme intermediate, which may exist in either a ferric or ferrous state (Fig. S1), as a potential target of H2S. In the enzymatic reaction, the macrocycle of verdoheme is cleaved by reductive O2 activation on its central iron (
      • Matsui T.
      • Nakajima A.
      • Fujii H.
      • Matera K.M.
      • Migita C.T.
      • Yoshida T.
      • Ikeda-Saito M.
      O2- and H2O2-dependent verdoheme degradation by heme oxygenase: Reaction mechanisms and potential physiological roles of the dual pathway degradation.
      ,
      • Matsui T.
      • Omori K.
      • Jin H.
      • Ikeda-Saito M.
      Alkyl peroxides reveal the ring opening mechanism of verdoheme catalyzed by heme oxygenase.
      ). Anomalously slow O2 binding on ferrous verdoheme is regarded as a major rate-determining step of HO to achieve the O2 dependence of its overall activity (
      • Matsui T.
      • Nakajima A.
      • Fujii H.
      • Matera K.M.
      • Migita C.T.
      • Yoshida T.
      • Ikeda-Saito M.
      O2- and H2O2-dependent verdoheme degradation by heme oxygenase: Reaction mechanisms and potential physiological roles of the dual pathway degradation.
      ,
      • Liu Y.
      • Ortiz de Montellano P.R.
      Reaction intermediates and single turnover rate constants for the oxidation of heme by human heme oxygenase-1.
      ). The other two O2 reactions with heme and hydroxyheme are not rate-limiting because of their high reactivity and/or affinity (
      • Migita C.T.
      • Matera K.M.
      • Ikeda-Saito M.
      • Olson J.S.
      • Fujii H.
      • Yoshimura T.
      • Zhou H.
      • Yoshida T.
      The oxygen and carbon monoxide reactions of heme oxygenase.
      ,
      • Matera K.M.
      • Takahashi S.
      • Fujii H.
      • Zhou H.
      • Ishikawa K.
      • Yoshimura T.
      • Rousseau D.L.
      • Yoshida T.
      • Ikeda-Saito M.
      Oxygen and one reducing equivalent are both required for the conversion of α-hydroxyhemin to verdoheme in heme oxygenase.
      ). Chemically, the verdoheme ring could be cleaved even by O2-independent hydrolysis (
      • Saito S.
      • Itano H.A.
      Verdohemochrome IXα: preparation and oxidoreductive cleavage to biliverdin IXα.
      ). Although the verdoheme hydrolysis proceeds only under physiologically irrelevant, strong alkaline conditions, we hypothesized that H2S efficiently promotes “thiolysis,” the hydrolysis-like ring cleavage (see Fig. 1A), because of its strong nucleophilicity even under physiological pH. As expected from the reaction design, the S-HO reaction is found to produce an unprecedented sulfur-containing biliverdin termed S-biliverdin (SBV). H2S reacts with ferric, but not ferrous, verdoheme with drastic modulation of the HO catalytic properties due to circumvention of the rate-limiting O2 reaction (Fig. 1).
      Figure thumbnail gr1
      Figure 1Novel heme degradation induced by H2S. A, initial design of H2S-induced branching of the HO reaction. Peripheral substituents are omitted for clarity. B, structures of SBV isomers. C, structures of BR and SBR isomers.

      Results

      Novel S-HO reaction under single-turnover condition

      Effects of H2S on HO catalysis were first examined under single-turnover conditions where ferric heme complexed with rat HO-1 was converted once into products. In the absence of H2S, reduction of ferric heme–HO-1 by its physiological redox partner, NADPH-cytochrome P450 reductase (CPR), resulted in an intricate absorption spectral change to eventually afford BV having intense visible absorption around 688 nm (Fig. 2A) (
      • Matsui T.
      • Furukawa M.
      • Unno M.
      • Tomita T.
      • Ikeda-Saito M.
      Roles of distal Asp in heme oxygenase from Corynebacterium diphtheriae, HmuO: a water-driven oxygen activation mechanism.
      ). Temporal emergence of sharp absorption at 641 nm is due to transient accumulation of CO-bound verdoheme (Fig. S1). In the presence of H2S, the starting heme complex showed a spectral shift typical for partial binding of H2S to the ferric heme iron (Fig. 2B) (
      • Bostelaar T.
      • Vitvitsky V.
      • Kumutima J.
      • Lewis B.E.
      • Yadav P.K.
      • Brunold T.C.
      • Filipovic M.
      • Lehnert N.
      • Stemmler T.L.
      • Banerjee R.
      Hydrogen sulfide oxidation by myoglobin.
      ). The heme degradation with H2S proceeded through distinct intermediate(s) absorbing around 754 nm to yield product(s) having broader absorption at 697 nm. Although H2S hardly affected amounts of CO produced through the heme degradation (Fig. S4D), HPLC analysis detected two new products other than BV in the presence of H2S (Fig. 2D), whose absorption spectra are similar to each other but different from that of BV (Fig. S4A). Further product analysis identified these two new products as SBV regioisomers, 1-SBV and 19-SBV (Fig. 1B). Positive mass signals of the new products were both found at 599.24 (Fig. S4C), consistent with the SBV formula ([C33H34O5N4S + H+]+; calculated m/z, 599.23). Isotope labeling experiments revealed incorporation of one each atom of oxygen and sulfur into the new products, whereas BV accepts two oxygen atoms from O2 at the cleavage site (Table S1). 1-SBV and 19-SBV standards synthesized according to a previous report (
      • Fuhrhop J.H.
      • Kruger P.
      1- or 19-methoxy-, 1- or 19-amino- and 1- or 19-thio-deoxybiliverdins.
      ) (Figs. S2 and S3) are indistinguishable from the two new products in terms of the HPLC retention time (Fig. 2D) and absorption spectra (Fig. S4, A and B). These results strongly suggest H2S-dependent ring cleavage as hypothesized.
      Figure thumbnail gr2
      Figure 2Effects of H2S on heme degradation by HO. A and B, absorption spectral changes of heme–HO-1 complexes upon reduction by CPR and NADPH without and with 100 μm H2S, respectively. Spectra were taken before reaction (black) with H2S (blue) and 15 min after addition of CPR (red). C, spectral changes in the presence of both CPR and BVR before and after reaction of heme–HO-1 (dashed and solid, respectively) without and with 200 μm H2S (black and red, respectively). Inset, relative absorbance increase without H2S at 460 nm (black) and with H2S at 530 nm (red). D, HPLC chromatograms for the reaction mixture in the presence of H2S and CPR (black) and the 1- and 19-SBV standards (blue and red, respectively). E, HPLC analysis of culture media of RAW264.7 cells treated with hemin (upper) and hemin and 2 mm H2S (middle) observed at 520 nm (black). Bottom traces are chromatograms obtained with mixed standards at 520 (black) and 450 nm (red). F, H2S dependence of the HO activity (black) and the SBR ratio (red) under catalytic condition. G, O2 dependence of the catalytic activity (black squares) and the SBR ratio (red squares) in the presence of 100 μm H2S of rat HO-1. Catalytic activity was also measured in the absence of H2S (open circle). Error bars represent S.E.

      Formation and properties of SBR

      In mammals, BV is rapidly reduced by biliverdin reductase (BVR) to a yellow pigment, bilirubin (BR; Fig. 1C and Fig. S4E). The single turnover reaction of the purified HO enzyme with additional BVR yielded BR having typical absorption around 462 nm (Fig. 2C). The SBV standards were also reduced by BVR to corresponding 1- and 19-SBR isomers (Fig. 1C), whose absorption peaks were observed around 530 nm to have distinct orange colors (Fig. S4, E–G). Structures of the SBR isomers were further confirmed by various product analyses (Fig. S5, A–C and Table S2). Due to the large red shifts of the absorption bands, the SBR formation under the single-turnover condition is easily visualized by an unusual shoulder around 530 nm (Fig. 2C). The single turnover rate slightly decreased in the presence of H2S as judged by absorbance increase either at 460 or 530 nm (approximately half; Fig. 2C).
      SBR is also produced by a macrophage cell line, RAW264.7. This cell line is reported to secrete BR into cell culture media when incubated with hemin (
      • Park J.H.
      • Oh S.M.
      • Lim S.S.
      • Lee Y.S.
      • Shin H.K.
      • Oh Y.S.
      • Choe N.H.
      • Park J.H.
      • Kim J.K.
      Induction of heme oxygenase-1 mediates the anti-inflammatory effects of the ethanol extract of Rubus coreanus in murine macrophages.
      ). Our HPLC analysis detected only BR as a product of the normal heme catabolism by RAW264.7 (Fig. 2E). Addition of H2S, however, resulted in the formation of the two SBR isomers as well as BR (Fig. 2E), assuring the progression of the S-HO reaction in mammalian cells.
      The SBR isomers share similar properties with BR as expected from their structural similarity (Fig. 1C). Highly hydrophobic SBR and BR require bovine serum albumin (BSA) for their solubilization in neutral buffers. BR is known to be excreted into bile upon glucuronidation in liver (Fig. S5D) (
      • Bosma P.J.
      • Seppen J.
      • Goldhoorn B.
      • Bakker C.
      • Oude Elferink R.P.
      • Chowdhury J.R.
      • Chowdhury N.R.
      • Jansen P.L.
      Bilirubin UDP-glucuronosyltransferase 1 is the only relevant bilirubin glucuronidating isoform in man.
      ). Both isomers of SBR were similarly converted by UGT1A1 to their mono- and diglucuronides (Fig. S5, E–G). BR is also proposed to be a potent antioxidant through a redox cycling with BV (Fig. S6A) (
      • Baranano D.E.
      • Rao M.
      • Ferris C.D.
      • Snyder S.H.
      Biliverdin reductase: a major physiologic cytoprotectant.
      ,
      • Stocker R.
      • Yamamoto Y.
      • McDonagh A.F.
      • Glazer A.N.
      • Ames B.N.
      Bilirubin is an antioxidant of possible physiological importance.
      ). Catalytic removal of radical species was suggested for BR and the SBR isomers in the 2,2′-azobis(2-amidinopropane) dihydrochloride (AAPH) assay (Fig. S6), implying their comparable potencies as antioxidants.

      Catalytic properties of the S-HO reaction

      The H2S effects on HO catalytic properties were examined in the presence of BVR (
      • Liu Y.
      • Ortiz de Montellano P.R.
      Reaction intermediates and single turnover rate constants for the oxidation of heme by human heme oxygenase-1.
      ). As shown in Fig. 2F, the product ratio of total SBR over all three products (%SBR) rose as the H2S concentration increased (EC50 = 34 μm). To our surprise, the catalytic activity of HO-1 was severely suppressed by H2S (Fig. 2F; IC50 = 48 μm) despite its small effect on the single turnover rate (Fig. 2C). Although the EC50 and IC50 values are similar at the basal condition ([CPR] = 100 nm), these parameters showed distinct behavior when the CPR concentration was lowered to 20 nm (EC50 and IC50 = 9.6 and 200 μm, respectively). This observation reveals different mechanisms for the SBR induction and HO inhibition by H2S. It should also be noted that %SBR was saturated at ∼45% under normal air. Incomplete formation of SBR is due, at least in part, to an unexpected action of O2 because %SBR increased with lowering O2 concentrations (Fig. 2G). O2 dependence of the catalytic activity was also largely affected by H2S (Fig. 2G). The normal HO reaction without H2S was slowed down at O2 concentrations less than 130 μm due to the rate-limiting reaction of the ferrous verdoheme intermediate with O2 (Fig. 1A) (
      • Matsui T.
      • Nakajima A.
      • Fujii H.
      • Matera K.M.
      • Migita C.T.
      • Yoshida T.
      • Ikeda-Saito M.
      O2- and H2O2-dependent verdoheme degradation by heme oxygenase: Reaction mechanisms and potential physiological roles of the dual pathway degradation.
      ,
      • Liu Y.
      • Ortiz de Montellano P.R.
      Reaction intermediates and single turnover rate constants for the oxidation of heme by human heme oxygenase-1.
      ). In contrast, very little O2 dependence was observed in the presence of H2S, consistent with circumvention of the slow O2-dependent ring cleavage (Fig. 1A).

      Ferric verdoheme as a branch point intermediate

      We finally examined reaction mechanism of the S-HO reaction. In our initial design, H2S was postulated to attack ferrous verdoheme (Fig. 1A); however, this intermediate showed negligible spectral change upon addition of H2S and CPR under anaerobic condition (Fig. S7A). Further O2 addition initiated the reaction but afforded only BR (Fig. S7A). Instead, more electrophilic ferric verdoheme, which was generated prior to the ferrous form (Fig. S1), was found to rapidly react with H2S. The H2S addition to ferric verdoheme diminished its characteristic absorption at 683 nm with appearance of featureless, “flat” absorption in a 600–800-nm range (Fig. 3A). Resulting intermediate X was reduced to yield a mixture of BR and SBR under normal air, whereas the SBR formation became dominant at lower O2 concentration (Fig. 3D), consistent with the O2 dependence of %SBR observed in the catalytic assay (Fig. 2G). Other HO intermediates generated prior to ferric verdoheme, including hydroxyheme (Fig. S1), did not react with H2S or only reversibly interacted to eventually afford the same intermediate X (Fig. S7, B–D). These results unambiguously identify ferric verdoheme as a branch point intermediate for the BV and SBV pathways of HO as shown in Fig. 4.
      Figure thumbnail gr3
      Figure 3Reactions of ferric verdoheme with H2S to afford SBR. A, spectral changes upon conversion of ferric heme–rat HO-1 (black) to ferric verdoheme (red) and then to the intermediate X with 100 μm H2S (blue). B, titration curve for reaction of ferric verdoheme with H2S fitted with a piecewise linear function. C, ESI-MS spectra (negative ion mode) of the intermediate X prepared with nonlabeled (black) and 34S-labeled H2S (red). D, absorption spectra of the intermediate X (dashed) and its reduced product by CPR and BVR (solid) at the indicated O2 concentrations. Inset, SBR ratios determined by HPLC analysis. E, pH dependence of apparent rate constants for the reaction between ferric verdoheme and H2S. F, anaerobic titration of X (black) and 1-SBV (red) with varied amounts of NADPH (2 reducing eq per molecule). SBR formation was estimated from the absorbance increase at 541 nm. Error bars represent S.E.
      Figure thumbnail gr4
      Figure 4Proposed mechanisms for the heme degradation in the presence of H2S. Peripheral substituents are omitted for clarity.
      Conversion of ferric verdoheme to the intermediate X required approximately 1 molar eq of H2S (Fig. 3B). Mass analysis also revealed incorporation of one sulfur atom into ferric verdoheme (Fig. 3C and Fig. S9A). Apparent rate constants for the formation of X show pH-dependent transition with a pKa value of 7.0 (Fig. 3E). This change is attributed to ionization of H2S (pKa = 7.0) because no significant spectral change was observed for ferric verdoheme in this pH range. The alkaline form (SH; 29 mm−1 s−1) was ∼300-fold more reactive than the acid form (H2S; 0.094 mm−1 s−1), indicating SH as a major reactant. This conclusion rationally explains the low reactivity of other biological thiols. Glutathione (GSH), l-cysteine, and homocysteine, which have higher pKa values (9.7, 8.4, and 8.9, respectively), showed only limited reactivity with ferric verdoheme (Fig. S8B). Moreover, these alkyl thiols did not convert ferric verdoheme to X but reduced it to the ferrous state, leading to the normal BV pathway (Fig. S8A and Fig. 4). Similar simple reduction was also observed for sodium polysulfides (Na2S2, Na2S3, and Na2S4) as shown in Fig. S8, C and D. Therefore, H2S is the specific thiol that induces SBV formation.

      Ring opening mechanism in the H2S-mediated reaction

      Under anoxic conditions, reduction of the intermediate X dominantly produced SBR (Fig. 3D), strongly suggesting an O2-independent hydrolysis-like mechanism for the SBV production (Fig. 4). In this mechanism, nucleophilic addition of SH to a β-pyrrole carbon atom of ferric verdoheme should be followed by deprotonation to cleave the macrocycle. A resulting Fe3+–SBV complex could be reduced by one electron to afford SBV with release of the ferrous iron. As expected from this scheme, the anoxic conversion of X to SBR consumed three electrons, two of which were utilized for reduction of SBV to SBR (Fig. 3F). In the presence of O2, the mechanism became more intricate to afford BV in addition to SBV. The BV formation from X required O2-induced extrusion of the sulfur atom. X itself has no reactivity with O2 as judged from the negligible O2 dependence of its absorption spectra (Fig. 3D). Instead, a reduced form of X may bind O2 on its central iron to liberate the SH moiety. This putative reaction yielded oxyferrous verdoheme, which is an intermediate proposed in the normal HO reaction (Fig. 4) (
      • Matsui T.
      • Nakajima A.
      • Fujii H.
      • Matera K.M.
      • Migita C.T.
      • Yoshida T.
      • Ikeda-Saito M.
      O2- and H2O2-dependent verdoheme degradation by heme oxygenase: Reaction mechanisms and potential physiological roles of the dual pathway degradation.
      ). The verdoheme regeneration from X is possible because pyridine extraction of X from the protein yielded a bispyridine complex of verdoheme (Fig. S9B). It should be also mentioned that one oxygen atom of O2 was inserted in the conversion of X to BV (Fig. S9C), consistent with the O2 activation on the verdoheme iron (
      • Matsui T.
      • Nakajima A.
      • Fujii H.
      • Matera K.M.
      • Migita C.T.
      • Yoshida T.
      • Ikeda-Saito M.
      O2- and H2O2-dependent verdoheme degradation by heme oxygenase: Reaction mechanisms and potential physiological roles of the dual pathway degradation.
      ).

      Discussion

      In this study, we report the new reaction pathway of mammalian HO induced by H2S. The gaseous signaling molecule drastically alters the reaction mechanism of HO not only to modulate the catalytic activity and its O2 dependence but also to produce the new sulfur-containing heme catabolites, 1- and 19-SBV isomers (Fig. 1). It is quite surprising that the classical HO enzyme, whose function and mechanism have been studied in detail over several decades, has an unidentified reaction pathway that is just now being revealed. Recently, noncanonical heme degradation has also been identified for a few bacterial enzymes; however, these enzymes are specifically designed to promote unusual reactions (
      • Nambu S.
      • Matsui T.
      • Goulding C.W.
      • Takahashi S.
      • Ikeda-Saito M.
      A new way to degrade heme: the Mycobacterium tuberculosis enzyme MhuD catalyzes heme degradation without generating CO.
      ,
      • Matsui T.
      • Nambu S.
      • Goulding C.W.
      • Takahashi S.
      • Fujii H.
      • Ikeda-Saito M.
      Unique coupling of mono- and dioxygenase chemistries in a single active site promotes heme degradation.
      ,
      • LaMattina J.W.
      • Nix D.B.
      • Lanzilotta W.N.
      Radical new paradigm for heme degradation in Escherichia coli O157:H7.
      ). We have succeeded in inducing the alternative reaction of the canonical HO enzyme by the rational selection of the small biological molecule to attack its reaction intermediate. H2S appears to have broad reactivities with many biological compounds. For example, heme is known to interact with H2S to cause catalytic oxidation of the sulfide (
      • Bostelaar T.
      • Vitvitsky V.
      • Kumutima J.
      • Lewis B.E.
      • Yadav P.K.
      • Brunold T.C.
      • Filipovic M.
      • Lehnert N.
      • Stemmler T.L.
      • Banerjee R.
      Hydrogen sulfide oxidation by myoglobin.
      ) and covalent modification of the heme periphery to generate sulfheme (
      • Johnson E.A.
      The reversion to haemoglobin of sulphhaemoglobin and its coordination derivatives.
      ). These reactions appear to be negligible upon induction of the S-HO reaction (Figs. 2B and 3B), probably due to the facile and specific reactivity of the HO intermediate with H2S. Our approach to target the enzyme intermediate thus allows identification of highly specific interactions and could be applied widely to search for hidden functions, especially of well-studied enzymes. Besides the SBV pathway, the HO reaction may branch into unidentified pathways at its numerous reactive intermediates (Fig. S1) by choosing other appropriate small molecules. Moreover, H2S could react with enzymes other than HO to bring out their unidentified reactions. To discover further unknown targets, possible effects of H2S should be extensively studied for reactions involving hydrolysis or a step replaceable with a hydrolysis-type reaction in addition to searching for electrophilic intermediates.
      We have almost fully elucidated the formation mechanism of SBV. The SH ion binds to ferric verdoheme and cleaves the macrocycle in an O2-independent manner. The only major ambiguity remaining is the nature of the observed intermediate X. The intermediate X generated in the reaction between ferric verdoheme and H2S can be either an SH–adduct of verdoheme or an Fe3+–SBV complex (Fig. 4), both of which match the mass data (Fig. 3C and Fig. S9A). The SH–adduct form well explains the O2-dependent SH extrusion, whereas ring closure of Fe3+–SBV seems not to occur easily. Nevertheless, the absorption spectrum of X, especially its flat absorption in the visible region, is very similar to that of the Fe3+–BV complex (Fig. 3A and Fig. S9D) (
      • Yoshida T.
      • Kikuchi G.
      Features of the reaction of heme degradation catalyzed by the reconstituted microsomal heme oxygenase system.
      ), strongly suggesting the Fe3+–SBV structure for X. The ring closure may proceed more easily than we expected to achieve equilibrium of the open and closed ring structures for X and/or its reduced form.
      Successful detection of SBR assures that the S-HO reaction can take place in mammalian cells (Fig. 2E). Nevertheless, we cannot specify the H2S concentration required for the SBV induction (EC50) in vivo. Our mechanistic analysis identifies ferric verdoheme as a branch point intermediate for the normal and SBV pathways (Fig. 4). Because the SH addition and reduction of ferric verdoheme are essentially irreversible, a major determinant of the EC50 value should be relative rates of the two competing reactions. Slower reduction by CPR hence results in the lowered EC50 value as shown for the in vitro assay. Some naturally occurring mutations of CPR are known to significantly suppress electron transfer to HO (
      • Marohnic C.C.
      • Huber Iii W.J.
      • Patrick Connick J.
      • Reed J.R.
      • McCammon K.
      • Panda S.P.
      • Martásek P.
      • Backes W.L.
      • Masters B.S.
      Mutations of human cytochrome P450 reductase differentially modulate heme oxygenase-1 activity and oligomerization.
      ), probably leading to hypersensitivity toward H2S. Hypoxia is also expected to decelerate the electron transfer by inducing liberation of HO from the endoplasmic reticulum (
      • Lin Q.
      • Weis S.
      • Yang G.
      • Weng Y.H.
      • Helston R.
      • Rish K.
      • Smith A.
      • Bordner J.
      • Polte T.
      • Gaunitz F.
      • Dennery P.A.
      Heme oxygenase-1 protein localizes to the nucleus and activates transcription factors important in oxidative stress.
      ). The cytosolic HO enzyme could interact only weakly with CPR remaining on the endoplasmic reticulum membrane. The SBV formation could be enhanced under hypoxia because of the slow reduction as well as the low O2 concentration. Nonmammalian HOs with greater diversity in structure and reactivity may have larger deviations in the H2S requirement to stimulate the S-HO reaction. Even non-HO–type heme-degrading enzymes involving ferric verdoheme in their catalysis are possible targets of H2S (
      • Uchida T.
      • Sekine Y.
      • Dojun N.
      • Lewis-Ballester A.
      • Ishigami I.
      • Matsui T.
      • Yeh S.R.
      • Ishimori K.
      Reaction intermediates in the heme degradation reaction by HutZ from Vibrio cholerae.
      ). Our finding clearly indicates the necessity to take the possible interplay between H2S and heme degradation into account in all related research areas.
      The new S-HO reaction could have large impacts on physiological functions of H2S and HO through drastic changes in both the product and catalytic activity of HO. Properties of the new heme catabolites have been examined mainly for their reduced form, SBR, which as well as BR should be the major form in mammalian cells. Although BR is crucial in mammals as a causative agent of jaundice and a potent antioxidant (
      • Baranano D.E.
      • Rao M.
      • Ferris C.D.
      • Snyder S.H.
      Biliverdin reductase: a major physiologic cytoprotectant.
      ), SBR shows solubility, glucuronidation potency, and redox properties similar to those of BR (Figs. S5, D–G, and S6). This observation implies that H2S does not greatly alter antioxidative functions of the HO reaction in mammals. Biological roles of SBV are yet to be examined, especially in biosynthesis of phytochromes and photoreceptor proteins in plants, bacteria, and fungi, which utilize BV or its derivatives as a pigment for detecting red and far-red light (
      • Ulijasz A.T.
      • Vierstra R.D.
      Phytochrome structure and photochemistry: recent advances toward a complete molecular picture.
      ).
      Our catalytic assay reveals the O2-dependent change of the SBR production ratio (%SBR) due to the O2-induced return to the BV pathway (Figs. 2G and 4). Considering the similar properties of BR and SBR, this product change itself may have a biological significance only when a receptor system specific to the new catabolite exists. More importantly, the O2 dependence of the HO activity was lost in the presence of H2S due to circumvention of the rate-limiting O2 activation by ferrous verdoheme (Figs. 2G and 4) (
      • Morikawa T.
      • Kajimura M.
      • Nakamura T.
      • Hishiki T.
      • Nakanishi T.
      • Yukutake Y.
      • Nagahata Y.
      • Ishikawa M.
      • Hattori K.
      • Takenouchi T.
      • Takahashi T.
      • Ishii I.
      • Matsubara K.
      • Kabe Y.
      • Uchiyama S.
      • et al.
      Hypoxic regulation of the cerebral microcirculation is mediated by a carbon monoxide-sensitive hydrogen sulfide pathway.
      ,
      • Matsui T.
      • Nakajima A.
      • Fujii H.
      • Matera K.M.
      • Migita C.T.
      • Yoshida T.
      • Ikeda-Saito M.
      O2- and H2O2-dependent verdoheme degradation by heme oxygenase: Reaction mechanisms and potential physiological roles of the dual pathway degradation.
      ,
      • Peng Y.J.
      • Makarenko V.V.
      • Nanduri J.
      • Vasavda C.
      • Raghuraman G.
      • Yuan G.
      • Gadalla M.M.
      • Kumar G.K.
      • Snyder S.H.
      • Prabhakar N.R.
      Inherent variations in CO-H2S-mediated carotid body O2 sensing mediate hypertension and pulmonary edema.
      ). This activity change is expected to have large impacts on gas signaling, namely the O2 sensing in the brain microvessels as well in as the carotid body. In these systems, HO-2, a constitutive isoform of HO, serves as a primary oxygen sensor, and its CO product is considered to transduce the O2 signal in concert with H2S (
      • Morikawa T.
      • Kajimura M.
      • Nakamura T.
      • Hishiki T.
      • Nakanishi T.
      • Yukutake Y.
      • Nagahata Y.
      • Ishikawa M.
      • Hattori K.
      • Takenouchi T.
      • Takahashi T.
      • Ishii I.
      • Matsubara K.
      • Kabe Y.
      • Uchiyama S.
      • et al.
      Hypoxic regulation of the cerebral microcirculation is mediated by a carbon monoxide-sensitive hydrogen sulfide pathway.
      ,
      • Williams S.E.
      • Wootton P.
      • Mason H.S.
      • Bould J.
      • Iles D.E.
      • Riccardi D.
      • Peers C.
      • Kemp P.J.
      Hemoxygenase-2 is an oxygen sensor for a calcium-sensitive potassium channel.
      ,
      • Peng Y.J.
      • Nanduri J.
      • Raghuraman G.
      • Souvannakitti D.
      • Gadalla M.M.
      • Kumar G.K.
      • Snyder S.H.
      • Prabhakar N.R.
      H2S mediates O2 sensing in the carotid body.
      ). A major H2S generator in brain is CBS (
      • Abe K.
      • Kimura H.
      The possible role of hydrogen sulfide as an endogenous neuromodulator.
      ), whose prosthetic heme group binds CO to directly inhibit the enzyme activity under normoxia (
      • Taoka S.
      • Banerjee R.
      Characterization of NO binding to human cystathionine β-synthase: possible implications of the effects of CO and NO binding to the human enzyme.
      ). CO is also reported to be a physiological inhibitor of cystathionine γ-lyase in the carotid body despite the absence of an apparent CO-binding site (
      • Yuan G.
      • Vasavda C.
      • Peng Y.J.
      • Makarenko V.V.
      • Raghuraman G.
      • Nanduri J.
      • Gadalla M.M.
      • Semenza G.L.
      • Kumar G.K.
      • Snyder S.H.
      • Prabhakar N.R.
      Protein kinase G-regulated production of H2S governs oxygen sensing.
      ). Under hypoxic condition, the HO activity decreases to relieve the CO inhibition of the H2S-generating enzymes. Consequently, hypoxia stimulates the H2S synthesis to enhance vasodilation in brain and neural activity of the carotid body through regulation of potassium channels (
      • Morikawa T.
      • Kajimura M.
      • Nakamura T.
      • Hishiki T.
      • Nakanishi T.
      • Yukutake Y.
      • Nagahata Y.
      • Ishikawa M.
      • Hattori K.
      • Takenouchi T.
      • Takahashi T.
      • Ishii I.
      • Matsubara K.
      • Kabe Y.
      • Uchiyama S.
      • et al.
      Hypoxic regulation of the cerebral microcirculation is mediated by a carbon monoxide-sensitive hydrogen sulfide pathway.
      ,
      • Peng Y.J.
      • Nanduri J.
      • Raghuraman G.
      • Souvannakitti D.
      • Gadalla M.M.
      • Kumar G.K.
      • Snyder S.H.
      • Prabhakar N.R.
      H2S mediates O2 sensing in the carotid body.
      ). The faster formation of H2S together with its slower decay through O2-dependent processes is expected to promote the O2-independent S-HO reaction under hypoxia (Figs. 2G and 4). This maintains the HO activity at a certain level even under highly hypoxic conditions to avoid a complete stop of CO synthesis and thus overproduction of H2S. An H2S concentration ceiling may be important to prevent respiratory disorder (
      • Peng Y.J.
      • Zhang X.
      • Gridina A.
      • Chupikova I.
      • McCormick D.L.
      • Thomas R.J.
      • Scammell T.E.
      • Kim G.
      • Vasavda C.
      • Nanduri J.
      • Kumar G.K.
      • Semenza G.L.
      • Snyder S.H.
      • Prabhakar N.R.
      Complementary roles of gasotransmitters CO and H2S in sleep apnea.
      ) and to reduce its toxicity. Likewise, under inflammatory conditions in which cytokines activate macrophages, the cells induce HO-1 (
      • Hayashi S.
      • Takamiya R.
      • Yamaguchi T.
      • Matsumoto K.
      • Tojo S.J.
      • Tamatani T.
      • Kitajima M.
      • Makino N.
      • Ishimura Y.
      • Suematsu M.
      Induction of heme oxygenase-1 suppresses venular leukocyte adhesion elicited by oxidative stress: role of bilirubin generated by the enzyme.
      ), which might protect against oxidative stress through the action of SBV and SBR under hypoxic conditions.
      Another striking effect of H2S is severe suppression of the HO catalytic activity (Fig. 2F), which is not explained by the mechanism shown in Fig. 4. Considering the weak inhibition of the single turnover reaction (Fig. 2C), interference by H2S appears to occur at a step prior to formation of the heme–HO complex. Under the catalytic conditions, excess hemin weakly binds to BSA supplemented to solubilize the BR and SBR products. The hemin–BSA complex exhibits a significant spectral change when incubated with H2S under hypoxic conditions but not under air, suggesting temporal reduction of hemin, O2 binding, and rapid auto-oxidation. This implies catalytic production of reactive oxygen species, which may inactivate the HO enzyme. The inhibitory effect of H2S in vivo might be distinct from our in vitro observation because it would largely depend on the status of “free heme” that interacts with various cellular factor(s).

      Experimental procedures

      Materials

      18O2 and 34S8 were purchased from Spectra Stable Isotope (18O content, >95%) and ISOFLEX (34S content, 99.9%), respectively. Liquid NH3 (20 ml) was added dropwise to 34S8 (100 mg, 2.94 mmol) and sodium (135 mg, 5.89 mmol) in a round-bottom flask at −78 °C. The reaction mixture was stirred at room temperature under an argon atmosphere until liquid NH3 entirely vaporized. The residue was dried under reduced pressure to afford Na234S (anhydrous) as pale yellow powder (233 mg, quantitative). Microsomes containing human UGT1A1 and reagents required for glucuronidation were obtained from BD Gentest. Preparation of rat HO-1, its hemin complex, human CPR, and BVR was carried out as described previously (
      • Matera K.M.
      • Zhou H.
      • Migita C.T.
      • Hobert S.E.
      • Ishikawa K.
      • Katakura K.
      • Maeshima H.
      • Yoshida T.
      • Ikeda-Saito M.
      Histidine-132 does not stabilize a distal water ligand and is not an important residue for the enzyme activity in heme oxygenase-1.
      ,
      • Fujii H.
      • Zhang X.
      • Tomita T.
      • Ikeda-Saito M.
      • Yoshida T.
      A role for highly conserved carboxylate, aspartate-140, in oxygen activation and heme degradation by heme oxygenase-1.
      ,
      • Sun D.
      • Sato M.
      • Yoshida T.
      • Shimizu H.
      • Miyatake H.
      • Adachi S.
      • Shiro Y.
      • Kikuchi A.
      Crystallization and preliminary X-ray diffraction analysis of a rat biliverdin reductase.
      ). Other chemicals and proteins were obtained from Wako, Oriental Yeast, Sigma-Aldrich, or ICN and used without further purification. RAW264.7 was purchased from the American Type Culture Collection (ATCC). The basal buffer used throughout this study was 0.1 m HEPES, pH 7.5, at 20 °C unless otherwise stated.

      Heme degradation by purified enzyme systems

      Single turnover reactions to yield BV/SBV were typically examined in the presence of 10 μm ferric heme–HO-1, 40 μm NADPH, 40 nm CPR, and varied amounts of H2S. Similar single turnover reactions for BR/SBR were supplemented further with 40 nm BVR and 10 μm BSA. Catalytic assays were conducted with 0.25 μm HO-1, 10 μm hemin, 200 μm NADPH, 100 nm CPR, 25 nm BVR, 15 μm BSA, 100 units/ml catalase, 10 units/ml superoxide dismutase, and varied concentrations of H2S. Initial rates were determined at 20 min to assure linear increase of the products. O2 dependence of the catalytic activity and 18O2 labeling of the products were examined in an anaerobic glove box (UNIlab, MBraun). Absorption spectral changes during the reaction were recorded by an Agilent 8453 or Shimadzu UV-1500 spectrophotometer.

      Product analysis of enzymatic reactions

      Heme catabolites were concentrated by solid-phase extraction with Supelclean LC-18 columns (Supelco) as described elsewhere (
      • Matsui T.
      • Nakajima A.
      • Fujii H.
      • Matera K.M.
      • Migita C.T.
      • Yoshida T.
      • Ikeda-Saito M.
      O2- and H2O2-dependent verdoheme degradation by heme oxygenase: Reaction mechanisms and potential physiological roles of the dual pathway degradation.
      ). The BR/SBR extraction was operated under minimum light to suppress their photoisomerization. Benzophenone was added as an internal standard at the end of the reactions for quantitative experiments. HPLC analyses of the extracted products were performed on a Shimadzu LC-20 HPLC system equipped with an SPD-M20A photodiode array detector at a flow rate of 1.0 ml/min. The BV/SBV products were separated on a Tosoh ODS-80Ts reversed-phase column (4.6 × 150 mm) with an isocratic elution of 70% methanol and 30% 0.1 m ammonium acetate (v/v). The BR/SBR analysis was performed typically on the ODS-80Ts column with a linear gradient from 75% methanol, 25% 20 mm triethylamine, and acetic acid (v/v), pH 5.0, to 85% methanol (v/v) over 15 min.
      ESI-MS spectra were measured on a Bruker micrOTOF-Q-II mass spectrometer equipped with an Agilent 1100 HPLC system at a flow rate of 0.2 ml/min. LC separation of BV/SBV was conducted on the ODS-80Ts column (2.0 × 150 mm) with an isocratic elution of 20% 0.1 m ammonium acetate and 80% methanol (v/v). BR/SBR and their glucuronides were analyzed with an Agilent ZORBAX Extend-C18 reversed-phase column (2.1 × 150 mm) using a linear gradient from 35% methanol and 65% 20 mm aqueous ammonia (v/v) to 50% methanol (v/v) over 20 min. Mass spectra of the intermediate X were measured by direct infusion at 3 μl/min of a reaction solution containing 3 μm heme–HO-1, 10 μm H2O2, and 20 μm H2S. Typical parameters for the MS measurements were as follows: end plate offset, −500 V; capillary, 4500 V; in-source collision-induced dissociation, 150 V; nebulizer gas, 0.4 bar; dry gas, 4.0 liters/min; dry gas temperature, 180 °C.
      CO was quantitated using an H64L variant of myoglobin having high CO affinity as reported earlier (
      • Nambu S.
      • Matsui T.
      • Goulding C.W.
      • Takahashi S.
      • Ikeda-Saito M.
      A new way to degrade heme: the Mycobacterium tuberculosis enzyme MhuD catalyzes heme degradation without generating CO.
      ). After completion of single turnover reactions of 4 μm heme–HO-1, solid sodium dithionite and then H64L myoglobin (final concentration, 6 μm) were added to record absorption spectra of H64L partially bound with CO. Subtraction of a spectrum of ferrous H64L without exogenous ligands generated difference spectra in which a positive peak indicates generation of the CO-bound form and a negative peak is due to disappearance of ferrous deoxy H64L.

      Synthesis of 1- and 19-SBV

      1- and 19-SBV isomers were synthesized as their dimethyl esters (total yields from BV, 22 and 15%, respectively) according to a previous report (
      • Fuhrhop J.H.
      • Kruger P.
      1- or 19-methoxy-, 1- or 19-amino- and 1- or 19-thio-deoxybiliverdins.
      ). 1H and 13C NMR of the dimethyl esters was measured in CDCl3 with JEOL JMN-LA400 and JMN-LA600 Fourier transform NMR spectrometers (Figs. S2 and S3). Chemical shifts were referenced internally to CDCl3. The SBV esters were hydrolyzed, purified, and weighed by a Sartorius ultra-microbalance SE2 to determine extinction coefficients (Fig. S4, F and G).

      Glucuronidation

      Approximately 20 μm SBV isomers were reduced by BVR to corresponding SBR isomers in the presence of 20 μm NADPH and 30 μm BSA. The freshly prepared SBR was glucuronidated at 37 °C by BD Supersomes UGT1A1 according to a protocol provided by the manufacturer. Aliquots of samples were analyzed by HPLC and ESI-MS.

      AAPH assay

      Each of 10 μm freshly prepared BR and SBR isomers was incubated at 37 °C with 50 mm AAPH in 0.1 m potassium phosphate, pH 7.5, containing 200 mm NaCl and 15 μm BSA with or without 100 μm NADPH and 50 nm BVR. Absorption spectral changes during the reaction were monitored at 468, 541, and 521 nm for BR, 1-SBR, and 19-SBR, respectively.

      Heme degradation by mammalian cells

      RAW264.7 cells were maintained in a 37 °C humidified incubator containing 5% CO2 and cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, penicillin-streptomycin, and glutamine. 9 × 106 cells were grown in a 75-cm2 cell culture flask for 24 h and then incubated with 20 μm hemin for 12 h to induce HO-1. For efficient removal of BR generated during the induction period, the cells and the flask were washed three times with the culture medium containing fetal bovine serum rather than PBS. Heme degradation was performed by incubating the cells in fresh medium (18 ml) containing 5 μm hemin and 2 mm H2S at 37 °C. The screw cap of the flask was closed during the reaction to suppress volatilization of H2S. The medium was recovered after 2-h incubation, centrifuged (3,000 rpm for 5 min), and supplemented with propiophenone as an internal standard. Further addition of a half-volume of saturated guanidine hydrochloride was followed by solid-phase extraction and HPLC analysis as noted above.

      Preparation of HO intermediates

      Ferric heme–HO-1 as purified was reduced by a slight excess of dithionite anaerobically in the glove box to afford ferrous deoxyheme–HO-1 to which 100 μl of air was bubbled to yield ferrous oxyheme–HO-1. Ferric hydroxyheme–HO-1 was generated in an anaerobic reaction of ferric heme–HO-1 with a slight excess of H2O2 (
      • Liu Y.
      • Moënne-Loccoz P.
      • Loehr T.M.
      • Ortiz de Montellano P.R.
      Heme oxygenase-1, intermediates in verdoheme formation and the requirement for reduction equivalents.
      ). Reaction of ferric heme–HO-1 with H2O2 under air yields ferric verdoheme–HO-1 (
      • Wilks A.
      • Ortiz de Montellano P.R.
      Rat liver heme oxygenase. High level expression of a truncated soluble form and nature of the meso-hydroxylating species.
      ). Alternatively, ferric verdoheme–HO-1 was generated upon oxidation of ferrous verdoheme–HO-1, which was prepared as described earlier (
      • Matsui T.
      • Nakajima A.
      • Fujii H.
      • Matera K.M.
      • Migita C.T.
      • Yoshida T.
      • Ikeda-Saito M.
      O2- and H2O2-dependent verdoheme degradation by heme oxygenase: Reaction mechanisms and potential physiological roles of the dual pathway degradation.
      ), by 3 molar eq of potassium ferricyanide (K3[Fe(CN)6]). The latter method enables completely anoxic preparation of ferric verdoheme. Ferric biliverdin–HO-1 was prepared from ferric heme–HO-1 by ascorbate-dependent heme degradation.

      Reaction of ferric verdoheme–HO-1 with H2S

      The ferric verdoheme complex was prepared by H2O2 immediately prior to use and used within 5 min after adding catalase to remove the residual peroxide. Reactions with alkyl thiols were performed in the presence of CO to suppress auto-oxidation of ferrous verdoheme. Kinetic analysis of ferric verdoheme–HO-1 with H2S was performed at 20 °C using a UNISOKU RSP-601 stopped-flow apparatus equipped with a built-in rapid scan spectrophotometer. The ferric verdoheme complex (3 μm) was mixed with equal volumes of H2S solution (final concentration, 50–200 μm). Observed rate constants determined from absorbance change at 683 nm exhibited good linear relationships with the H2S concentrations to afford apparent reaction rate constants (kapp) at varied pH. pH dependence of the kapp values (Fig. 3E) is well-fitted by assuming an acid-base transition of H2S (pKa 7.0). Buffers used for the kinetic analysis were: 0.1 m citrate, pH 6.0–7.0; phosphate, pH 6.5–7.5; HEPES, pH 7.0–8.0; and Tris-HCl, pH 7.5–8.5.

      Product formation from X

      The intermediate X was prepared anaerobically from ferrous verdoheme as noted above. The reductive titration to SBR was performed under anoxic condition while monitoring absorbance increase at 541 nm. Formal two-electron reduction of SBV to SBR was confirmed for the stable 1-SBV isomer. To examine O2 dependence of the product distribution, the anoxic X was mixed with air-saturated buffer to fill the screw-top cuvette. Products were quantitated by HPLC analysis. For isotope labeling of BV generated through O2-dependent reaction of X, the intermediate was prepared with a slight excess of nonlabeled H2O2 and air, which was followed by addition of catalase and bubbling with excess 18O2.

      Author contributions

      T. M., M. S., and M. I.-S. conceptualization; T. M. and M. I.-S. funding acquisition; T. M., R. S., K. S., Y. T., M. I., Y. N., and T. H. investigation; T. M. and M. I.-S. writing-original draft; T. M. and M. I.-S. project administration; M. S. and M. I.-S. writing-review and editing.

      Acknowledgments

      A portion of this work was supported by metabolomics platform established by JST, ERATO, Suematsu Gas Biology (completed in March 2015). We thank Drs. C. S. Raman, Emma Raven, and Sofia M. Kapetanaki for their helpful comments.

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