A Multistep Mechanism Proposed for the - ACS Publications

Article Cite This: Biochemistry XXXX, XXX, XXX−XXX

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Mechanistic Insights into the Activation of Soluble Guanylate Cyclase by Carbon Monoxide: A Multistep Mechanism Proposed for the BAY 41-2272 Induced Formation of 5‑Coordinate CO−Heme Ryu Makino,*,† Yuji Obata,† Motonari Tsubaki,‡ Tetsutaro Iizuka,§ Yuki Hamajima,† Yasuyuki Kato-Yamada,† Keisuke Mashima,† and Yoshitsugu Shiro*,∥ †

Department of Life Science, College of Science, Rikkyo University, Nishi-ikebukuro 3-34-1, Toshima-ku, Tokyo 171-8501, Japan Department of Chemistry, Graduate School of Science, Kobe University, Kobe, Hyogo 657-8501, Japan § RIKEN Harima Institute/Spring8, 1-1-1 Kouto, Mikazuki-cho, Sayo-gun, Hyogo 679-5148, Japan ∥ Graduate School of Life Science, University of Hyogo, 3-2-1 Kouto, Kamigori-cho, Ako-gun, Hyogo 678-1297, Japan ‡

S Supporting Information *

ABSTRACT: Soluble guanylate cyclase (sGC) is a heme-containing enzyme that catalyzes cGMP production upon sensing NO. While the CO adduct, sGC−CO, is much less active, the allosteric regulator BAY 41-2272 stimulates the cGMP productivity to the same extent as that of sGC−NO. The stimulatory effect has been thought to be likely associated with Fe-His bond cleavage leading to 5coordinate CO−heme, but the detailed mechanism remains unresolved. In this study, we examined the mechanism under the condition including BAY 41-2272, 2′-deoxy-3′-GMP and foscarnet. The addition of these effectors caused the original 6-coordinate CO−heme to convert to an end product that was an equimolar mixture of a 5- and a new 6-coordinate CO−heme, as assessed by IR spectral measurements. The two types of CO−hemes in the end product were further confirmed by CO dissociation kinetics. Stopped-flow measurements under the condition indicated that the ferrous sGC bound CO as two reversible steps, where the primary step was assigned to the full conversion of the ferrous enzyme to the 6-coordinate CO−heme, and subsequently followed by the slower second step leading a partial conversion of the 6coordinate CO−heme to the 5-coordinate CO−heme. The observed rates for both steps linearly depended on CO concentrations. The unexpected CO dependence of the rates in the second step supports a multistep mechanism, in which the 5coordinate CO−heme is led by CO release from a putative bis-carbonyl intermediate that is likely provided by the binding of a second CO to the 6-coordinate CO−heme. This mechanism provides a new aspect on the activation of sGC by CO.

S

Scheme 1

oluble guanylate cyclase (sGC) is the best-characterized NO receptor involved in cell−cell signal transduction pathways associated with several critical physiological processes.1−5 Mammalian sGC with a heterodimeric (αβ) structure binds a stoichiometric amount of heme via a weak Fe−proximal His bond between the heme-iron and His105 of the β-subunit.6 The binding of NO to the unliganded ferrous heme is very rapid and stimulates the guanosine 3′,5′-cyclic monophosphate (cGMP) production from guanosine 5′-triphosphate (GTP) over several hundred-folds by triggering a series of intramolecular signaling from the heme-domain to the catalyticdomain of the enzyme. The reaction of the ferrous sGC with NO was summarized in Scheme 1. The primary product in the reaction with NO is a 6-coordinate NO adduct, which coordinates NO at the distal site of the heme with very high affinity.7,8 Subsequently, the bound NO cleaves the weak Fe− His bond by pulling up the heme-iron into heme plane (called negative trans effect), which leads a distal 5-coordinate NO adduct with NO at the distal site of the heme as a sole ligand.7,8 Unexpectedly, the rates of the formation of the 5-coordinate © XXXX American Chemical Society

Received: December 11, 2017 Revised: February 12, 2018

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DOI: 10.1021/acs.biochem.7b01240 Biochemistry XXXX, XXX, XXX−XXX

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Biochemistry

substrate GTP or its analogue GTP-γ-S gave valuable information regarding the activation mechanisms by YC-1/ BAY 41-2272.21−23 These results indicated that the population of 5-coordinate CO−heme was markedly increased by combined treatment of YC-1/BAY 41-2272 and GTP/GTP-γS, and was likely sufficient to support the high-level activity of the sGC−CO complex. In contrast, the addition of GDP or ATP has no or little effect on the population of 5-coordinate CO−heme, and therefore, it is reasonable to assume that the enhanced formation of 5-coordinate CO−heme is mediated by occupation of both recognition sites for nucleotide base and triphosphate moiety of the nucleotides.21,22 Furthermore, a recent Raman study using the site directed variants of sGC provided the strong support that the appearance of the 5coordinate CO−heme does correlate to the high-level activation.24 These results together strengthened the possibility that the 5-coordinate CO−heme serves as a key species in the BAY/YC-1-induced activation of the sGC−CO complex. Our previous kinetic study indicated that 2′-d-3′-GMP acts as a potent purine-site (P-site) inhibitor by binding to the catalytic site of the BAY−sGC−CO complex together with pyrophosphate (PPi). While the P-site inhibitor complex [BAY−(sGC−CO)−2′-d-3′-GMP−PPi complex] could not be isolated as stable species, it was stabilized when foscarnet was used as an analogue of PPi in the presence of Mn2+.25 The resultant P-site inhibitor complex [BAY−(sGC−CO)−2′-d-3′GMP−foscarnet] which is schematically illustrated below, is substantially equivalent to the BAY−(sGC−CO)−GTP complex, in respect that 2′-d-3′-GMP and foscarnet occupied the nucleotide and phosphate recognition sites, respectively.

NO adduct were found to increase with increasing NO concentrations.8 This anomalous kinetic behavior was thought to result from the binding of a second NO to the proximal side of the 6-coordinate NO−heme with forming a transient bisnitrosyl heme species (bis-NO−heme), which quickly lost the original distal NO leading to a proximal 5-coordinate NO− heme as the end product. Consequently, both the formation of the 6-coordinate NO−heme and the conversion of it to the proximal 5-coordinate NO−heme are NO dependent. Indeed, a recent mechanistic study supports the interconversion of the NO coordination from the distal to the proximal heme face.9 The unique proximal 5-coordinate NO−heme has been identified in crystal structures of the NO complex of cytochrome c′10 and of the heme-nitric oxide/oxygen binding domain (H-NOX) from Shewanella oneidensis.11 In contrast to the NO binding, the affinity of sGC for CO is an anomalously low (200−300 μM), as compared to that of other high spin ferrous hemoproteins with coordination of a proximal histidine.12−14 This predicts that there may be an unfavorable Fe-proximal base orientation or steric constraints of the proximal base to the in-plane movement of the Fe atom required for the CO binding, as pointed out for other hemoproteins.15 The binding of CO yielded a stable 6coordinate CO−heme with stimulation of the cyclase activity by ∼5-fold relative to that of the basal state of the enzyme.16 The slight stimulation in response to CO was suggested to be achieved by the conversion of a very small fraction of 6coordinate CO−heme to an active 5-coordinate CO−heme.12 This hypothesis is proposed based on the analogy with the model for stimulatory mechanism of sGC by NO. In this connection, of most interest is the finding that an antiplatelet coagulating reagent YC-1 [(3-(5′-hydroxymethyl-3′-furyl)-1benzylindazole)] or BAY 41-2272 [5-cyclopropyl-2-(1-((2fluorophenyl)methyl)pyrazolo(3,4-b)pyridin-3-yl)pyrimidin-4amine] remarkably stimulates the cyclase activity of sGC−CO to nearly the same extent as sGC−NO.17−19 This provided an important clue in revealing the mechanistic route of the formation of the 5-coordinate CO−heme, as described below.

In the present study, it was confirmed that the state of the CO−heme in this P-site inhibitor complex is a mixture of a 5and a 6-coordinate CO−heme in a single conformational state, which were reversibly redistributed with temperature. The temperature-dependent behavior provided a beneficial clue enabling the mechanistic analyses of the 5-coordinate CO− heme formation by kinetical examinations. The basis for this approach comes from the following assumption: the two types of CO−heme species is likely to be in equilibrium at a constant temperature, in which a fraction of the 6-coordinate CO−heme may be driven to the 5-coordinate CO−heme by shifting the equilibrium upon increasing CO concentration. The stoppedflow kinetic measurements provided definite evidence for the multistep binding of CO to sGC, in which the 5-coordinate CO−heme was led from a fraction of 6-coordinate CO−heme. The proposed mechanism in this paper provides new mechanistic insights into the activation of sGC by CO.

The correlation between the coordination state of CO−heme and the enhanced enzymatic activity upon exposure to YC-1 or BAY 41-2272 has been extensively studied by vibrational spectroscopies18−21 which allow the determination of coordination number of CO−heme by a π-back-bonding correlation of νC−O vs νFe−CO frequencies. These studies, as expected, provide clear spectroscopic evidence for the formation of a 5coordinate CO−heme as well as 6-coordinate CO−heme in several conformationally distinct states. Although the formation of the 5-coordinate CO−heme was thought to be associated with the activation of sGC catalysis, the level was apparently low to support a remarkable increase in the catalytic activity by the allosteric stimulators. We noted that these vibrational measurements were exclusively carried out in the absence of the substrate, and did not necessarily reflect the actual state that served under catalytic conditions including GTP. Subsequent resonance Raman studies focusing on the effects of the

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EXPERIMENTAL PROCEDURES Reagents. 2′-d-3′-GMP was purchased from Sigma-Aldrich Japan (Tokyo, Japan), and further purified using a high performance liquid chromatography (a C18 column). YC-1 and BAY 41-2272 were purchased from ALEXIS (San Diego, CA). Other chemicals, purchased from Wako Chemicals Co. (Tokyo, Japan), were of the highest commercial grade. CO and NO

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CO binding, the stock CO solution was diluted to desired concentrations with an anaerobic buffer. In stopped-flow experiments described above, anaerobic conditions were maintained by adding a catalytic amount of catalase and glucose oxidase in the presence of 0.5 mM glucose. The buffer used was 50 mM Hepes (pH 7.4) containing 50 mM NaCl and 4 mM Mn2+, unless otherwise stated. Under the conditions to require BAY 41-2272 or YC-1, DMSO was included in the buffer at a final concentration of 2% (v/v), to maintain the solubility. The rate constants for the CO or the NO binding were determined by a single or a double exponential fitting to the kinetic data using GraphPad Prism (version 4.03) or IgorPro (version 5.03). The gastight luer-lock syringes were used as sample reservoirs to maintain anaerobic conditions throughout stopped-flow experiments. The rapid scan timeresolved spectra were measured under the condition to minimize photodissociation of the bound CO by reducing the intensity of the incident white light using a filter cutting UV light below 330 nm.

gases were obtained from Takachiho Chemicals Co. (Tokyo, Japan) Enzyme Purification. Fresh bovine lung (5 kg) was minced and homogenized using a Waring blender in 15 L of 50 mM potassium phosphate buffer, pH 7.4 containing a mixture of protease inhibitors (1 mM phenylmethanesulfonyl fluoride, 1 mM benzamidine, and 1 mM ethylenediaminetetraacetic acid) and 55 mM β-mercaptoethanol. Protease inhibitors and βmercaptoethanol were included in all the buffers throughout the purification unless stated otherwise. The protocol for the enzyme purification was essentially the same as described previously,20 except for the use of Mimetic Orange 1 affinity absorbent (Sigma-Aldrich, St Louis, MO) in place of Cibacron Brilliant Red 3B-A affinity absorbent.26 The purified enzyme preparations supplemented with 5% glycerol and 5 mM DTT were stored in liquid nitrogen until they were use. Spectral Measurements. The ferrous CO complex of sGC was prepared by adding the fully reduced sGC to the buffer solution equilibrated with CO gas at 1 atm in a septum-sealed anaerobic cuvette. After the headspace of the cuvette was filled with 1 atm of CO, the optical absorption spectra were recorded on a PerkinElmer Lambda 18 spectrophotometer (PerkinElmer Japan, Tokyo) equipped with a cuvette holder thermostatically controlled by thermomodule elements. IR spectra were measured by a PerkinElmer Spectral One FTIR spectrophotometer (PerkinElmer Japan, Tokyo) with a mercury− cadmium-telluride detector. The data were collected at 2 cm−1 resolution. The cell had CaF2 windows with a light path length of 0.1 mm. The desired temperature of the cell was maintained by a circulating water bath, a Julabo F13. The temperature of the IR cell holder was measured with a thermocouple. The band areas of the each IR spectral component were determined by deconvolution with Gaussian fitting function (IgorPro version 5.03). The residuals between the observed and simulated spectra were illustrated by dotted lines in the corresponding figures. The buffer was consisted of 50 mM Hepes (pH 7.4) containing 50 mM NaCl, 5% (v/v) ethylene glycol, 2.5 mM dithiothreitol, and 5 mM Mn2+. In the cases to assess the effects of BAY 41-2272 or YC-1, DMSO was added to maintain their solubility at a final concentration of 4% (v/v). Stopped-Flow Measurements. The binding of CO to the ferrous enzymes was analyzed by a DX-18MV stopped-flow spectrophotometer, equipped with a photodiode array detector (Applied Photophysics, Leatherhead, UK). The combination rates of the enzymatic heme with CO and NO were determined by mixing the anaerobic ferrous sGC solution with anaerobic solutions containing a desired amount of CO or NO using the stopped-flow apparatus at 15 °C. NO gas was purified by bubbling through 0.5 M KOH solution to remove the higher nitrogen oxides. NO saturated buffer solution was prepared by equilibrating the degassed buffer solution with the purified NO gas at 1 atm in a Schlenk tube with tightly sealed rubber septum cap. The concentration of NO was determined by spectrophotometric titrations of the known amount of ferrous myoglobin with an aliquot of the NO stock solution via a gastight syringe under anaerobic conditions. In the NO determinations, the ferrous myoglobin was prepared by adding 1.2-fold excess NADPH in the presence of catalytic amounts of ferredoxin and its reductase. Stock solutions of CO were prepared by equilibrating the degassed buffer solution with a pure CO gas at 1 atm. The concentration of dissolved CO was assumed to be 1.35 mM CO at 15 °C and at 1 atm. For measurements of the

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RESULTS Optical and Infrared Spectra of CO Adducts of sGC. Optical spectrum of the sGC−CO adduct exhibited a sharp Soret peak at 424 nm, and the coordination structure was identified to be 6-coordinate CO−heme with proximal His by an X-ray absorption spectroscopy (EXAFS).26 The peak position of the CO adduct is unchanged with or without metal cofactor including Mg2+ or Mn2+. Effects of allosteric activators YC-1 and its analogue BAY 412272 on the coordination state of sGC−CO−heme have been extensively examined by a resonance Raman spectroscopy.18−24 As has been noted in those studies, the Fe-CO moiety displayed the complicated Raman spectra that were composed of several νFe−CO vibrational modes both in the absence and the presence of YC-1/BAY 41-2272. In the present study aiming to understand the molecular basis of the 5-coordinate CO−heme formation, a relative population of each component was carefully estimated by using FTIR, since the band intensities of the IR spectra ensure a reliable estimation, in contrast with those of resonance Raman spectra which are selectively enhanced depending on excitation-wavelength. The high quality FTIR spectra of the sGC−CO complex measured in the presence of Mn2+ could be deconvoluted to three IR bands with the peak positions at 1987.5, 1982, and 1968 cm−1 using a Gaussian fitting function (Figure 1). These νC−O modes had their origin of 6-coordinate CO−heme.19−23 Each of the νC−O modes at 1987.5 and 1982 cm−1 has the corresponding νFe−CO Raman mode (i.e., 1987.5/473 and 1982/489 cm−1 pairs), but the νFe−CO vibration paired with the 1968 cm−1 νC−O mode is currently unidentified. Replacing Mn2+ with Mg2+ has little effect on these IR spectral signatures (data not shown). A sharp, intense band at 1987.5 cm−1 diminished its amplitude upon elevating temperature, along with an increase in the intensities of other IR bands. This temperature-dependent change was fully reversible and merely resulted in a redistribution of the three components without showing detectable changes in their peak positions (inset in Figure 1A). The addition of a nearly stoichiometric amount of BAY 412272 markedly altered the FTIR spectra with an appearance of new IR bands at 1964 and 1971 cm−1 (Figure 2). The observed four νC−O modes redistributed upon changing temperature (inset of Figure 2A). The 1964 and 1971 cm−1 bands have been C


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Biochemistry

Figure 1. FTIR spectra of sGC−CO complex. (A) Infrared νC−O stretching at 15 °C, (B) νC−O stretching at 25 °C, and (C) νC−O stretching at 32 °C, with no addition. In the inset of figure (A), the relative band areas of each νC−O mode that were estimated by Gaussian curve fitting, were plotted against temperature. The enzyme concentration was 170 μM as heme. The buffer included 5 mM Mn2+.

Figure 2. FTIR spectra of sGC−CO complex in the presence of BAY 41-2272. (A) Infrared νC−O stretching measured at 15 °C, (B) νC− O stretching at 25 °C, and (C) νC−O stretching at 32 °C, in the presence of 180 μM BAY 41-2272. In the inset of figure (A), the relative band areas of each νC−O mode were plotted against temperature. The enzyme concentration was 160 μM as heme. The buffer included 5 mM Mn2+.

assigned to the νC−O mode of a 5-coordinate CO−heme and of a new 6-coodinate CO−heme, respectively, by π-back bonding correlation of νC−O vs νFe-CO frequencies.19 The present result provides definite evidence for the formation of the new 6-coordinate CO−heme, which was not obviously characterized in the previous report19 owing to the lower spectral resolution. Although the other two components located at 1981 and 1987.5 cm−1 are observed also in the absence of BAY 41-2272 (Figure 1), the addition of BAY 412272 significantly narrowed the 1981 cm−1 band and slightly broadened the 1987.5 cm−1 band. These observations reinforced that the four νC−O modes were from the Febound CO of the BAY-bound form of the sGC−CO complex. The tight binding of BAY 41-2272 to sGC was also supported by the kinetic finding that the addition of a nearly equimolar amount of BAY 41-2272 led to the full activation of the enzyme catalysis (supplemental data of ref 27), and the affinity was estimated to be less than 5 μM using the truncated forms of sGC from Manduca sexta.28 More simple IR spectrum was given by further addition of 2′d-3′-GMP and foscarnet. In the presence of Mn2+, foscarnet is exclusively used in place of PPi, because of the limited solubility of the Mn2+−PPi complex. As shown in Figure 3, the resultant IR spectrum is composed of a major sharp overlapping νC−O band and a very small band at 1987 cm−1. The latter 1987 cm−1 band can be virtually ignored due to its small contribution at all temperatures investigated. The 1964 and 1971 cm−1 bands comprised in the overlapping νC−O band were assigned to the νC−O mode of 5- and 6-coordinate CO−heme, respectively,19,25 both of which were in a conformational state with a

single Fe-CO orientation. The νC−O peak position of the 5coordinate CO−heme is located in the lower wavelength region than that of the 6-coordinate CO−heme, as reported in the previous heme-model study.29 The relative contribution of the two major components at 1964 and 1971 cm−1 significantly changed with temperature: the 5-coordinate CO−heme (1964 cm−1 band) gained and the 6-coordinate CO−heme (1971 cm−1 band) diminished the intensity upon lowering temperature (inset in Figure 3A). The fractional population of the 5-coordinate CO−heme was the highest at 15 °C in a temperature range examined (∼45% in population) (inset in Figure 3A). Even under this condition leading to a high level of 5-coordinate CO−heme, both CO− heme components are fully reversible upon temperature changes [compare (a) with (d) in Figure 3A]. Thus, the simultaneous binding of 2′-d-3′-GMP and foscarnet may restrict the conformational freedom of heme pocket, thereby converting the four Fe-bound CO species of the BAY-bound sGC−CO to the only two types of CO−heme species (i.e., a 5and a 6-coordinate CO−heme). The clear temperaturedependent behavior differed from that in the presence of BAY alone: with BAY alone, the level of the 1964 cm−1 mode was almost independent of temperature (inset in Figure 2A), being consistent with the previous result.19 Direct optical spectroscopic measurements of the sGC−CO complex in an IR cell indicated that the Soret peak position of the sGC−CO complex was slightly but significantly blue-shifted by lowering temperature (Figure 3B). The similar spectral shift D


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Figure 4. CO binding kinetics and optical spectra in the presence of BAY 41-2272, 2′-d-3′-GMP and foscarnet. (A) Rapid scan measurements after mixing of the ferrous sGC (2.4 μM) with 1350 μM CO solution at 15 °C under anaerobic conditions. In the upper panel, spectra taken in the time range to 1020 ms after mixing were illustrated, and in the lower panel, spectra taken from 2.6 to 138 s after mixing were shown. Insets in the upper and the lower panel showed the visible spectra collected at 1020 ms and 138 s, respectively, in the expanded scales. These spectra were an average of four independent spectra taken at indicated times. (B) Optical absorption spectra of the sGC−CO complex at 15 °C. In the upper panel, the optical spectrum of the effector-free sGC−CO, and in the lower panel, the spectrum in the presence of 80 μM BAY 41-2272, 500 μM 2′-d-3′-GMP and 500 μM foscarnet is shown. These spectra were recorded by an ordinary spectrophotometer. The buffer included 5 mM Mn2+ in (A) and (B).

Figure 3. FTIR spectra of sGC−CO complex in the presence of BAY 41-2272, 2′-d-3′-GMP, and foscarnet. (A) Infrared νC−O stretching bands in the presence of 180 μM BAY 41-2272, 410 μM 2′-d-3′-GMP, and 680 μM foscarnet were measured at 15 °C (a), 25 °C (b), and 32 °C (c). The infrared νC−O stretching was remeasured at 15 °C (d) after the measurement at 32 °C to ensure the reversibility. In the inset, the relative band areas of each νC−O mode were plotted against temperature. The enzyme concentration was 160 μM as heme. (B) Optical spectra of the sample in an IR cell (CaF2 window) of 0.1 mm light path, which was prepared in (A), measured at 15 and 32 °C. The illustrated optical spectra are an average of 5 scans. The buffer included 5 mM Mn2+ in (A) and (B).

For comparison, we briefly summarized the effects of YC-1 on the νC−O bands of sGC−CO. The addition of YC-1 caused an appearance of new νC−O modes at 1964 and 1973 cm−1 (Figure S1 of the Supporting Information). The 1964 and 1973 cm−1 modes had been assigned to a 5- and a 6-coordinate CO− heme,20 respectively, and their peak positions were essentially the same as those of the BAY-treated sGC−CO, except for slightly higher frequency shift by 2 cm−1 in the YC-1 bound 6coordinate CO-heme. Elevating temperature intensified the 1964 and 1973 cm−1 modes, probably by enhancing the affinity of YC-1.25 The binding of 2′-d-3′-GMP to the YC-1 bound form of sGC−CO intensified the 1964 and 1973 cm−1 bands with a concomitant loss of the 1987 cm−1 band (Figure S2 of the Supporting Information). The peak height of the 1964

of the Soret band associated with temperature changes was also observed in the lower concentration of the enzyme (∼1.0 μM) (data not shown). The 418 nm spectral species may comprise a considerable contribution of the 5-coordinate CO−heme, as suggested by the temperature-dependent behavior of FTIR spectra. On the other hand, the effector-free sGC−CO exhibited the 424 nm Soret peak (upper panel in Figure 4B), which was unaffected by temperature changes (data not shown). The correlation of the Soret peak position with the Fe-CO coordination structure is described later. E


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Biochemistry Table 1. Kinetic Parameters for CO Binding to sGC 5-coordinate-His ↔ 6-coordinate-CO kon (M−1 s−1) a

Exp1 Exp2b Exp3c Exp4d

(4.35 ± 0.15) × 10

4

(5.20 ± 0.20) × 104 (3.26 ± 0.12) × 104

6-coordinate-CO ↔ bis-CO-heme

koff (s−1)

kon (M−1 s−1)

koff (s−1)

4.6 ± 0.7 ∼3.1 5.2 ± 0.2 9.0 ± 0.8

27.4 ± 3.5

0.006 ± 0.002

a In the presence of BAY 41-2272, 2′-d-3′-GMP, and foscarnet. bNO trapping in the presence of BAY 41-2272, 2′-d-3′-GMP, and foscarnet. cIn the presence of BAY 41-2272. dNo addition.

cm−1 band was prominently intensified by the further addition of foscarnet (Figure S3 of the Supporting Information), in which the relative band area increased upon lowering temperature (inset in Figure S3A of the Supporting Information). This trend in temperature dependence is consistent with that of the BAY-treated enzyme (inset in Figure 3A). Overall, the peak positions of the individual νC−O IR mode in the presence of YC-1 are comparable to those in the presence of BAY 41-2272, but the level of 5-coordinate CO−heme is noted to be significantly lower in the case of YC1. In addition to these results, the production of 5-coordinare CO−heme absolutely requires an allosteric activator including YC-1 or BAY 41-2272, because neither of 2′-d-3′-GMP nor foscarnet generates 5-coordinate CO−heme (e.g., Figure S4 of the Supporting Information). The FTIR measurements described above indicated that the temperature-dependent changes in the IR spectra were a consequence of the displacement of the equilibrium between the 5- and 6-coordinate CO−heme. As stated earlier, we inferred that the equilibrium between the two components is achieved at a fixed temperature, and shifts toward the formation of the 5-coordinate CO−heme upon increasing CO concentrations. The interpretation motivated us to solve the mechanistic route for the 5-coordinate CO−heme formation using a stopped-flow approach. Optical Spectroscopic and Kinetic Characterization of CO Binding Reactions. In an initial attempt of mechanistic analyses, the binding of CO to the unliganded ferrous sGC were examined by a stopped-flow method in the absence of effectors at 15 °C. The reactions were monitored at two wavelengths to allow separate detection of 5- or 6-coordinate CO−heme formation. This is because the former species can be detected at 419 nm and the latter at 423 nm, when a 5coordinate CO−heme is sequentially produced from a 6coordinate CO−heme as described in the latter part of this section. When the formations of sGC−CO were followed after mixing with excess CO, both of time courses at 419 and 423 nm conformed to a single exponential kinetics with essentially the same rates (inset in Figure S5A of the Supporting Information). This confirms the sole conversion of the ferrous heme to the 6-coordinate CO−heme in the absence of effectors. The kobs value of the CO association is a first-order with respect to CO concentration (Figure S5B of the Supporting Information). The bimolecular association rate constant (kon) and the dissociation rate constant (koff) were calculated from the slope and the y-intercept in the plot of the kobs vs CO concentration, respectively: (3.26 ± 0.12) × 104 M−1 s−1 and 9.0 ± 0.8 s−1 (Table 1 and Figure S5B of the Supporting Information). This finding indicates that the three conformers detected by IR measurements are in a rapid equilibrium, and interconvert on a time scale faster than that of stopped-flow conditions.

The formation of 5-coordinate CO−heme was examined in the presence of BAY 41-2272, 2′-d-3′-GMP and foscarnet at 15 °C. Intriguingly, the rapid scan measurements clearly indicated the sequential formation of two spectral species after mixing with excess CO (Figure 4A). On the time scale of ∼1 s after mixing, the unliganded ferrous enzyme fully converted to a CO−heme species with a Soret peak at 423 nm giving a set of isosbestic points (upper panel in Figure 4A). The Soret band of the CO−heme species is symmetric, and does not contain significant signature of the unliganded ferrous heme (upper panel in Figure 4A and inset in Figure 5D). The peak positions of this species in the Soret and visible regions essentially agree with those obtained by an ordinary spectrophotometer in the absence of BAY 41-2272 (upper panel in Figure 4B). On the longer reaction time of a few minutes, the spectrum cleanly changed to a final spectrum with a Soret peak at 418.5 nm (lower panel in Figure 4A). There were no further spectral changes. The optical spectrum of the CO−heme end product was virtually identical to that of the CO complex obtained by an ordinary spectrophotometer after incubating with BAY, 2′-d3′-GMP and foscarnet (lower panel of Figure 4B). The single-wavelength measurements using a stopped-flow apparatus gave definite evidence for the formation of the two spectral species in the reaction with CO. Rapid absorbance increases at 419 and 423 nm occurred immediately after mixing, and then were followed by the slower absorbance changes which occurred with the opposite change in the amplitude at 419 and 423 nm (Figure 5A). Such slow absorbance changes were never detected in the reaction of the effector-free sGC with CO (Figure S5A of the Supporting Information). These single-wavelength measurements support the formation of two types of CO−heme species in Figure 4A, and also confirm that rapid scan data does not result from a relatively strong light intensity required for data acquisition by a photodiode array detector. The fast and slow phases measured at 419 and 423 nm can be separately fitted to a single exponential equation under the various CO concentrations (Figure 5A, and Figure S6A of the Supporting Information). In the fast phase, the observed CO association rate constants (kobs) measured at 419 nm agree with those measured at 423 nm, and linearly increase depending on the CO concentration as shown in the plot of kobs vs CO concentration (Figure 5B). The bimolecular association rate (kon) and the dissociation rate constant (koff) were determined to be (4.35 ± 0.15) × 104 M−1 s−1 and 4.6 ± 0.7 s−1 from the slope and the y-intercept in the plot, respectively (Table 1). The koff value is roughly the same as those obtained in the absence and the presence of BAY 41-2272 (koff of 9.0 ± 0.8 and 5.2 ± 1.2 s−1, respectively; Table 1 and Figure S5B of the Supporting Information). The initial CO−heme product, which is characterized by the slow association and the fast dissociation rates, could be assigned to 6-coordinate CO−heme. In addition F


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Biochemistry

band, as compared with those of the 6-coordinate CO−heme in the fast phase. In addition, the observed spectral features essentially agreed with those reported in the presence of BAY 41-2272 and GTP-γ-S,30 and also with those in the presence of BAY, 2′-d-3′-GMP and foscarnet in open cuvette measurements (lower panel of Figure 4B). The Soret band of the second CO adduct at 418.5 nm is slightly broader and less symmetric than that of the initial CO adduct, certainly indicating that the second CO adduct comprises a considerable contribution of the 423 nm absorption species (inset in Figure 5D). This slightly asymmetric feature is supported by the current FTIR investigations indicating that the CO−heme end product is composed of a mixture of 5- and 6-coordinate CO− heme in a nearly equal population. Subtraction of the contribution of 6-coordinate CO−heme from the spectrum of the CO−heme end product yielded a spectrum with a sharp, symmetric Soret band at 417 nm (Figure 5D). One may argue a possibility that the 417 nm species may correspond to 6coordinate CO-heme with a very weak Fe-His bond. To our knowledge, such 6-coordinate CO complex has not been reported even for model-hemes. Although the argument for the formation of the putative 6-coordinate CO-heme cannot be ignored, comparison between the present optical spectral and vibrational data allowed us to assign the 417 nm species to 5coordinate CO-heme. As was stated above, the stopped-flow traces in the slow phase follows a single exponential function (Figure 5A). Of particular importance, the rates of the slow phases monitored at 419 and 423 nm were slightly but significantly accelerated upon increasing CO concentration (compare Figure 5A with Figure S6A of the Supporting Information). The observed rates for the slow phases exhibited a linear dependence on CO concentrations, giving the bimolecular CO association rate constant of 27.4 ± 3.5 M−1 s−1 and the y-intercept of 0.006 ± 0.002 s−1 (Figure 5C and Table 1). The latter rate at the intercept was assigned to the CO dissociation rate constant from the putative bis-CO−heme, based on the proposed mechanism for the formation of bis-NO−heme.9 To test the concentration dependency of the effectors included, the CO binding reactions were measured under the condition with 2-fold amounts of foscarnet and 2′-d-3′-GMP in the presence of 685 μM of CO. An increase in the concentration of the effectors has little effect on the CO binding rate, and the rate constant fell on the line both in the plots of the fast and the slow phases. These kinetic results together suggest that the rate limiting step for the slow phase requires an additional binding of CO molecule. We remark here the reason why the present experiments are exclusively performed in the presence of Mn2+. We tested which of Mn2+ or Mg2+ maximizes the level of the 5-coordinate CO−heme in the reaction with CO. For instance, as shown in Figure S6B of the Supporting Information, the absorbance changes in the slow phase are small and less evident under the condition including Mg2+, in comparison to those in the case of Mn2+. The minor absorbance changes appear to be a result of a small population of the 5-coordinate CO−heme or relatively rapid interconversion between 5- and 6-coordinate CO−heme. Thus, the use of Mn2+ markedly simplifies interpretation of data, and seems to be a key selection allowing the clear and separate detection of 5-coordinate CO−heme. CO Dissociation from sGC−CO End Product. Next, we analyzed the CO dissociation from the sGC−CO end product by mixing with excess NO to examine whether the end product comprised two types of CO−heme species in the presence of

Figure 5. (A) Stopped-flow traces of the CO binding reactions were recorded at 423 nm (a) and 419 nm (b) after mixing of the ferrous sGC (1.1 μM) with CO (685 μM) at 15 °C. In insets, the time courses at 419 and 423 nm in the fast phase were shown in the expanded scales. Open circles denote theoretical points fitted to a single exponential function. The residuals from curve fittings are plotted above the time course curves (dashed curves). (B) The kobs for CO combination in the fast phase was plotted as a function of CO concentrations. (C) The kobs for CO combination in the slow phase was plotted against CO concentrations. In these experiments, the sample and the reference solutions in gastight syringes included 80 μM BAY 41-2272, 500 μM 2′-d-3′-GMP and 500 μM foscarnet. (D) The arithmetic spectrum corresponding to 5-coordinate CO−heme was indicated by the solid line. The dotted and dashed lines indicated the optical spectrum collected at 1020 ms in the fast and at 138 s in the slow phase, respectively. The buffer included 5 mM Mn2+ in all the cases.

to these kinetic results, it is noted that the initial CO adduct exhibits the peak positions at 423, 541, and 570 nm, comparable to those of 6-coordinate CO−heme in the absence of the effectors, reported by us (424, 541, and 570.5 nm in the upper panel in Figure 4B) and others.30 Based on the kinetic and optical spectral properties, we concluded that the initial CO adduct was 6-coordinate CO−heme complex. The second CO adduct, the end product in the slow phase exhibited the peak positions at 418.5, 538, and 570.5 nm (lower panel of Figure 4A): the Soret band was blue-shifted by 4.5 nm, along with 3 nm blue-shift of the β-band and a sharpening of αG


Article

Biochemistry BAY, 2′-d-3′-GMP and foscarnet. The NO trapping method is ordinarily the most reliable way to estimate the CO dissociation rate of CO−hemoproteins, because the rate limiting step for NO displacement is the dissociation of CO, and NO rapidly and tightly binds to the enzymatic heme preventing CO rebinding. In fact, the rates of the formation of the 6-coordinate NO−heme (∼108 M−1 s−1) and of its successive conversion to the 5-coordinate NO−heme (4.2 × 105 M−1 s−1) in the reaction of the ferrous sGC with NO at 15 °C are much faster than that of the CO release. The reaction of the sGC−CO end product with NO was measured by the stopped-flow photodiode array detector under the condition including BAY 412272, 2′-d-3′-GMP and foscarnet. The data clearly indicated the transformation of the sGC−CO end product to 5coordinate NO complex characterized by the peak position at 399 nm, showing the displacement of CO by NO (Figure 6A). Single-wavelength stopped-flow traces measured at 400 and 419 nm displayed two discrete phases, a fast phase completed within a few seconds and a slow phase on time scales of minutes (Figure 6B, and Figure S7 of the Supporting Information). The first-order rate constants for the fast phase were determined by a double-exponential curve fitting on milliseconds to minutes time scales. The rate constants of the fast phase thus obtained were independent of NO concentrations (koff of ∼3.1 s−1; Table 1 and Figure 6C), and were essentially the same as the koff 4.6 s−1, the CO dissociation rate constant in the presence of the effectors (Figure 5B). Hence, we assigned that the fast phase was the CO release step from the 6-coordinate CO−heme. In contrast, the rate constants for the slow phase (Figure 6B, and Figure S7 of the Supporting Information) show a hyperbolic dependence on NO concentrations (Figure 6D). This result suggests rate limitation by a step prior to NO binding, which is presumably assigned to the dissociation step of the coordinated CO from the 5-coordinate CO−heme, as discussed later.

Figure 6. CO dissociation from the CO−heme end product measured by NO trap. The CO−heme end product was prepared in a 2.5 mL gastight syringe containing the reaction mixture of 685 μM CO, 80 μM BAY 41-2272, 500 μM 2′-d-3′-GMP, and 500 μM foscarnet. (A) The reaction mixture containing the end CO−heme product (2.1 μM) was mixed with the anaerobic buffer containing 210 μM NO, 80 μM BAY 41-2272, 500 μM 2′-d-3′-GMP, and 500 μM foscarnet at 15 °C. The time-resolved rapid scan spectra were an average of four independent spectra taken at indicated times. (B) Stopped-flow trace measured at 400 nm after mixing 2.4 μM sGC with 126 μM NO. In the inset, the time course monitored at 400 nm is shown in the expanded scale. Other conditions are the same as those described in (A). Open circles denote theoretical points fitted to a double exponential function. The residuals from curve fittings are plotted above the time course curves (dashed curves). (C) The kobs in the fast phase under the various CO concentrations was plotted as a function of NO concentrations. (D) The kobs in the slow phase was plotted against NO concentrations. The buffer included 5 mM Mn2+ in all the cases.

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DISCUSSION The formation of the 5-coordinate CO−heme is specific to sGC treated with YC-1 or BAY 41-2272, and unknown for other hemoproteins except for heme-copper oxidase.31,32 Among resonance Raman studies focusing on the effects of YC-1/BAY 41-2272, it was particularly noticed that GTP and its analogue GTP-γ-S markedly affected the νFe−CO Raman mode of 5-coordinate CO−heme,22,23 as well as the νFe−NO Raman mode of 5-coordinate NO−heme.33 However, these nucleotides are likely to be metabolized. To avoid the risk, we employed a stable nucleotide 2′-d-3′-GMP, and revealed that the simultaneous binding of 2′-d-3′-GMP and foscarnet to the catalytic site of the BAY−sGC−CO complex markedly intensified the level of the 5-coordinate CO−heme by mimicking the role of GTP. The CO binding measurements by rapid mixing under the above conditions exhibited distinct biphasic kinetics, in which the slower phase corresponded to the partial conversion of the 6-coordinate CO−heme to the 5coordinate CO−heme. These results imply that the binding of CO to the heme in sGC weakens the Fe-proximal His bond via interaction between BAY 41-2272 and nucleotide binding sites. Since CO, unlike NO, shows a positive trans effect strengthening the Fe-proximal His bond, the effect is perhaps canceled by several factors including proximal steric constraints. In this connection, it is noted that the binding of t-butyl isocyanide with a larger size in comparison with CO or NO does not lead 5-coordinate heme in the presence of BAY 41-

2272, 2′-d-3′-GMP and foscarnet (data not shown). Thus, the resultant formation of 5-coordinate heme with an axial exogenous ligand seems to be mediated by small ligand, being diffusible to the proximal side of heme. Recent study using photoaffinity labeling technique provided strong support that BAY binds at the interface of the two HNOX domains in the α and β subunits.34 However, the binding of BAY 41-2272 at the site has little effect on the association rate for CO (Table 1) and also for t-butyl isocyanide with larger size (data not shown), implying that the BAY binding caused no significant conformational changes preventing or enhancing H


Article

Biochemistry Scheme 2

Scheme 3

be a consequence of deformation of sGC−CO heme, and is likely to be a basis of fast CO dissociation (∼9 s−1) from species f). The binding of YC-1/BAY 41-2272 produces the geometrical change with net planarization of the CO−heme [species (b)], as suggested by resonance Raman spectroscopic measurements.22−24 The effect appears to be associated with a decrease in the CO dissociation rate, while the difference is small (Table 1). Although there is no structural information for the 5-coordinate CO−heme [species (d) or (e)], it is known that the 5-coordinate CO model-heme adopts planar heme geometry.29 In Scheme 2, an excess CO is required to cause the partial conversion of the 6-coordinate CO−heme [species (b)] to the 5-coordinate CO−heme [species (d) or (e)]. As shown in the concerted model (Scheme 2), a second CO could bind to the proximal site of the 6-coordinate CO−heme with forming the species c). In the case of the binding of sGC with excess NO, the possible role of residue(s) including cysteine as an another target of the second NO binding site is argued.42 This possibility can be excluded for the CO binding, because CO is inert molecule unlike NO, and the ability to modify cysteine residue has not been reported. The rates of the conversion to the 5-coordinate CO−heme depend on the concentration of CO (bimolecular rate constant, 27.4 M−1 s−1). This unpredicted CO dependence suggests that the rate limiting step for this conversion requires transient binding of a second CO. Otherwise, the CO concentration dependence may not be observed. While bis-CO−heme is identified in a model heme complex,29 it may be difficult to be found in hemoproteins. In the subsequent step, CO release from the putative bis-CO−heme leaves either of the distal (d) or the proximal 5-coordinate CO−heme (e), in which the balance of affinities may determine which of two CO ligands dissociates from the heme-iron. Similar mechanism has been proposed to account for the controlled NO binding to cytochrome c′ in terms of a “balance affinities mechanism”.43 Although it has been pointed out that the formation of the proximal 5-coordinate CO−heme is less likely,22,23 there is no

the access of the exogenous ligands to the iron atom from the distal side of the heme.15 The effect of YC-1/BAY 41-2272 leading to changes in the CO binding properties of sGC may be achieved by an alternative mechanism. It is noted that rearrangement of heme geometry is suggested to play a key role in the regulation of the iron reactivity in prokaryotic H-NOX (or called SONO35) domains. In some of these H-NOX domains, the deviation from planarity of the heme in unliganded ferrous state is prominent, and maintained by a nonbonded contact of a Pro residue conserved in the proximal side of the heme.11,36−39 The functional significance of the heme distortion was tested using the site-directed mutant of H-NOX domain. For instance, it is reported that the replacement of Pro with Ala in T. tengcongensis H-NOX caused the heme flattening with the change in the heme reactivity for O2.37 However, binding data of CO and NO for the proline mutant are not measured and the reported νFe−CO for the proline mutant is the same as the wild type H-NOX. The significance of the heme distortion in biological function was also reported for other H-NOX.40 Furthermore, the Pro residue appears to participate in the cGMP productivity of sGC.24 Thus, the structural changes in the heme appear to be responsible to the function of the HNOX proteins. In Scheme 2, we summarized the reactions of sGC with CO and the effects of BAY 41-2272, and briefly discussed in relation to the heme structure which was proposed based on the results obtained by EXAFS and resonance Raman studies. As shown in Scheme 2, the heme-iron of unliganded ferrous sGC [species (a) in Scheme 2] exhibited significant out-ofplane displacement toward the proximal His26 (0.56 Å for sGC vs 0.48 Å for deoxy Mb). The out-of-plane Fe-displacement may cause deformation of porphyrin core, although it does not necessarily ensure the distortion of the pyrrole rings of heme.41 It is also noted that sGC−CO [species (f)] exhibited a considerable out-of-plane Fe-displacement toward the proximal side26 (0.13 Å for sGC−CO vs 0.02 Å for myoglobin−CO with a planar heme). The unusual displacement of heme-iron may I


Article

Biochemistry firm evidence to indicate which of a distal or proximal 5coordinate CO−heme is favorably formed. Therefore, the possibility for proximal CO binding in the 5-coordinate CO− heme cannot be ruled out. Effects of BAY 41-2272 on CO binding dynamics of sGC have been extensively investigated by laser-flash photolysis methods in a wide range from picosecond to second.30,44,45 As noted in these works, the binding of BAY 41-2272 caused the significant amplitude changes in the absorbance on the time ranges of picosecond to nanosecond, and the effect was augmented by further addition of GTP-γ-S.45 Under the conditions including BAY and GTP-γ-S, it is reasonable to assume that the photodissociation of the 5-coordinate CO− heme immediately forms 4-coordinate heme (e.g., species c in Scheme 3). Nonetheless, Yoo and co-workers45 ascribe the large amplitude change to a geminate rebinding of CO to the ferrous 5-coordinate heme retaining proximal His residue. This interpretation seems to propose the possibility that after flash photolysis, the ferrous 5-coordinate heme with the distal His residue was predominantly formed prior to the geminate CO recombination to the 4-coordinate heme. Although similar mechanisms are proposed from time-resolved resonance Raman measurements of sGC−CO in the presence of YC-1 and GTP,22,23 further experiments will be required to uncover detailed effects of BAY 41-2272 responsible for the formation of 5-coordinate CO−heme. The CO dissociation from the sGC−CO end product showed the distinct biphasic time course (Figure 6B, and Figure S7 of the Supporting Information). The observed rates for the slow phase exhibited the hyperbolic dependence on NO concentrations, giving the rate of 0.048 s−1 at an infinite concentration of NO (Figure 6D). This slow CO dissociation rate may be limited by either rate of interconversion between 5and 6-coordinate CO−hemes, or of CO escape from the 5coordinate CO−heme. We initially thought that CO is released after the formation of the 6-coordinate CO−heme which is in equilibrium with the 5-coordinate CO−heme. In this mechanism, the rate of the conversion step limits the overall rate of the CO dissociation, and must be comparable to or less than 0.006 s−1, as estimated from Scheme 2. The experimental results indicating that the observed rate constant (0.048 s−1) is larger than the predicted rate constant, do not support the CO dissociation route via 6-coordinate CO−heme. The most straightforward and the simplest model to rationalize the CO dissociation kinetics is to predict the transient formation of a 4-coordinate heme in the process. This competition mechanism is illustrated in Scheme 3, where the key feature in this scheme proposes the formation of 4coordinate heme (species c). For the reaction, kobs is given by eq 1.46 The terms in the equation were defined in Scheme 3. Equation 1 is reduced to a simple hyperbolic equation shown by eq 2, given the rate of NO dissociation is assumed to be very slow. When this equation was kobs =

k −COk+NO(NO) + k −NOk+CO(CO) k+NO(NO) + k+CO(CO)

kobs =

k −COk+NO(NO) k+NO(NO) + k+CO(CO)

(2)

Thus, the rate for the slow phase appears to correspond to the CO dissociation rate from the 5-coordinate CO−heme. To our knowledge, this study is the first report to represent a multistep mechanism for the formation of 5-coordinated CO− heme in the reaction of sGC with CO, in which the participation of a second CO in the formation of bis-CO− heme species provides the basis for the CO dependence in the conversion of the 6-coordinate CO−heme to the 5-coordinate CO−heme. The mechanism is proposed based on the findings only under the restricted experimental conditions, and therefore does not necessarily ensure the formation route of the 5-coordinate CO−heme under the catalytic conditions including GTP. Nevertheless, it is conceivable that the 5coordinate CO−heme could serve as an physiologically relevant species in sGC/CO signaling pathway as proposed in olfactory neurons,47 if endogenous compounds mimicking the function of YC-1/BAY 41-2272 may exist and potentiate the responsiveness of the enzymatic heme to endogenously occurring level of CO. Further efforts will be required to define that the YC-1/BAY 41-2272-induced activation by CO is mediated by the 5-coordinate CO−heme under the catalytic conditions.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.biochem.7b01240. Infrared absorption spectra of sGC−CO with YC-1; infrared absorption spectra of sGC−CO with YC-1 and 2′-d-3′-GMP; infrared absorption spectra of sGC−CO with YC-1, 2′-d-3′-GMP and foscarnet; infrared absorption spectra of sGC−CO with 2′-d-3′-GMP; CO binding kinetics to ferrous sGC in the presence of BAY 41-2272 and its absence; stopped-flow measurements of CO association to ferrous sGC in the presence of effectors;and stopped-flow measurements of CO dissociation kinetics from the CO-heme end products in the presence of effectors (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*(R.M.) Tel: 81-042-747-0658. E-mail: [email protected]. jp. *(Y.S.) Tel: 81-0791-58-0325. E-mail: [email protected]. jp. ORCID

Ryu Makino: 0000-0002-6890-3797 Funding

This work was supported by the Strategic Research Foundation Grant-aided Project for Private Universities (S1201003) from the Ministry of Culture, Education, Sports, Science and Technology of Japan.

(1)

applied to the observed rates in the slow phase, the limiting rate at an infinite NO concentration corresponds to k−CO. This is given as the y-intercept in the double reciprocal plot, 1/kobs vs 1/[NO].

Notes

The authors declare no competing financial interest. J


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ACKNOWLEDGMENTS The authors would like to thank Mieko Taketsugu for helpful technical assistance.

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ABBREVIATIONS sGC, soluble guanylate cyclase;; NO, nitric oxide; CO, carbon monoxide; GTP, guanosine 5′-triphosphate; 2′-d-3′-GMP, 2′deoxy-3′-guanosine monophosphate (2′-deoxyguanosine 3′monophosphate); cGMP, guanosine 3′,5′-cyclic monophosphate; GTP-γ-S, guanosine 5′-(γ-thio)triphosphate;; PPi, pyrophosphate; FTIR, Fourier transformed infrared spectroscopy; IR, infrared spectroscopy; DMSO, dimethyl sulfoxide; Hepes, 4-(2-hydroxyethyl)-1- piperazineethanesulfonic acid; Kd, dissociation constant; kon, association rate constant; koff, dissociation rate constant; νFe−CO, stretching vibration of Fe-CO unit; νC−O, stretching vibration of heme-bound C−O; H-NOX, heme-nitric oxide/oxygen; EXAFS, extended X-ray absorption fine structure

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