Reaction Intermediates of Nitric Oxide Synthase from Deinococcus

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Reaction Intermediates of Nitric Oxide Synthase from Deinococcus radiodurans as Revealed by Pulse Radiolysis; Evidence for Intramo-lecular Electron Transfer from Biopterin to Fe-O Complex II

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Yuko Tsutsui, Kazuo Kobayashi, Fusako Takeuchi, Motonari Tsubaki, and Takahiro Kozawa Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.7b00887 • Publication Date (Web): 10 Jan 2018 Downloaded from http://pubs.acs.org on January 10, 2018

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Biochemistry

Reaction Intermediates of Nitric Oxide Synthase from Deinococcus radiodurans as Revealed by Pulse Radiolysis; Evidence for Intramolecular Electron Transfer from Biopterin to FeII-O2 Complex Yuko Tsutsui,‡ Kazuo Kobayashi*‡, Fusako Takeuchi,† Motonari Tsubaki,⁑ and Takahiro Kozawa‡ ‡

The Institute of Scientific and Industrial Research, Osaka University, Mihogaoka 8-1, Ibaraki

Osaka 567-0047, Japan †

Institute for Promotion of Higher Education, 1-2-1 Tsurukabuto, Nada-ku, Kobe, Hyogo 657-

8501, Japan ⁑

Graduate School of Science, Department of Chemistry, 1-1 Rokkodai-cho, Nada-ku, Kobe,

Hyogo 657-8501, Japan Corresponding Author *E-mail: [email protected] Telephone : +81-6-6879-8501

ACS Paragon Plus Environment

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ABBREVIATIONS H4B, (6R)-5,6,7,8-tetrahydro-L-biopterin; H4F, (6S)-5,6,7,8-Tetrahydrofolic acid; NOS, Nitric oxide synthase; NHA, N-hydroxy-L-arginine; DrNOS, Deinococcus radiodurans nitric oxide synthase; eaq-, hydrated electron;


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ABSTRACT: Nitric oxide synthase (NOS) is a cytochrome P450-type mono-oxygenase that catalyzes the oxidation of L-arginine (Arg) to nitric oxide (NO) through a reaction intermediate N-hydroxy-L-arginine (NHA). The mechanism underlying the reaction catalyzed by NOS from Deinococcus radiodurans was investigated using pulse radiolysis. Radiolytically-generated hydrated electrons reduced the heme iron of NOS within 2 µs. Subsequently, ferrous heme reacted with O2 to form a ferrous-dioxygen intermediated with a second-order rate constant of 2.8 × 108 M-1 s-1.

In the tetrahydrofolate (H4F)-bound enzyme, the ferrous-dioxygen

intermediate was found to decay an another intermediate with a first-order rate constant of 2.2 × 103 s-1. The spectrum of the intermediate featured an absorption maximum at 440 nm and an absorption minimum at 390 nm. In the absence of H4F, this step did not proceed, suggesting that H4F was reduced with the ferrous-dioxygen intermediate to form a second intermediate. The intermediate further converted to the original ferric form with a first-order rate constant of 4 s-1. A similar intermediate could be detected after pulse radiolysis in the presence of NHA, although the intermediate decayed more slowly (0.5 s-1). These data suggested that a common catalytically active intermediate involved in the substrate oxidation of both Arg and NHA may be formed during catalysis. In addition, we investigated the solvent isotope effects on the kinetics of the intermediate after pulse radiolysis. Our experiments revealed dramatic kinetic solvent isotope effects on the conversion of the intermediate to the ferric form, of 10.5 and 2.5 for Arg and NHA, respectively, whereas the faster phases were not affected. These data suggest that the proton transfer in DrNOS is the rate-limiting reaction of the intermediate with the substrates.


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Nitric oxide (NO) is an ubiquitous signaling molecule involved in a diverse array of cellular processes associated with regulating the activities of the cardiovascular, nervous, and immune systems.1-6 NO is synthesized by nitric oxide synthase (NOS), which catalyzes the O2-dependent conversion of L-arginine (Arg) to L-citrulline via two consecutive reactions with Nω-hydroxy-Larginine (NHA) as a stable intermediate (Figure 1).7 NOS from mammals is composed of an Nterminal oxygenase domain containing a cysteine-ligated heme and ((6R)-5,6,7,8-tetrahydro-Lbiopterin (H4B) cofactors, a C-terminal reductase domain that binds flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD), and an intervening calmodulin binding region.8-12 Dioxygen binding and NO synthesis take place at a P450-type heme in the oxygenase domain; the electrons are provided by NADPH via two flavin moieties in the reductase domain. During catalysis, electron transfer proceeds from NADPH through FAD and FMN in NOS reductase to the oxygenase domain. Several bacterial species have been identified as harboring NOS-like enzymes in their genome.13 Bacterial NOS enzymes contain a single domain with high sequence homology to the oxygenase domain of mammalian NOS but without the associated reductase domain.14,

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Reductase partners for the bacterial NOSs have not yet been identified. NOSs from Deinococcus radiodurans, Bacillus subtilis, Staphylococcus aureus, and Bacillus anthracis have been shown to produce NO in vitro.16-18 The conservation of nearly all key residues involved in substrate and cofactor binding among the mammalian and bacterial NOSs suggests a similar mechanism of NO production.14 In D. radiodurans, another reduced pteridine, tetrahydrofolate (H4F), supports NO synthesis.15, 19 The NOS reaction sequence has been modeled based on the cytochrome P450-type monooxygenase sequential reactions (Figure 2). The reduction of FeIII-heme (1) to FeII-heme (2) via reductase enables dioxygen-binding and the formation of a ferrous heme-dioxygen complex (FeII-O2) (3). The enzyme-bound biopterin acts as an electron donor to the heme-dioxygen complex to form the peroxo-ferriheme (4). The iron-peroxo species (4) can react with a substrate


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directly, or it can accept a proton to form the hydroperoxo-ferriheme (5). Next, O-O bond scission occurs to generate water and an iron-oxo species, compound I (6), which is thought to hydroxylate the substrate. These intermediates, (4) and (5), have been identified only at cryogenic temperatures upon formation by radiolytic reduction methods.20-22 Compound I of NOS (6) was observed via peroxyacid treatment,23 during which substrate oxidation of NHA was suggested as an alternative cycle in the peroxidase-type activity.24, 25 In a solution comprising the enzymatic reaction at ambient temperatures, the stages (4)→(5) →(6), as shown in Figure 2, are sufficiently fast that no active species have been detected. In these steps, electron transfer from the pterin cofactor to the heme is the rate-limiting step for substrate oxidation.26 The two reaction steps (Figure 1) from Arg to NHA and then from NHA to citrulline, appear to follow a distinct mechanism mediated by a compound I type of ferryl intermediate and a peroxy intermediate, respectively.27-31 The mechanism, however, remains elusive, as none of the putative intermediates have been identified experimentally under solution conditions. A powerful approach to investigating unstable intermediates involves a pulse radiolysis technique in which a hydrated electron (eaq-) rapidly reduces the FeIII heme of various hemoproteins. 32-42 The pulse radiolysis method has several advantages to characterize reaction mechanisms. First, it permits the extremely rapid donation of a single electron to a metal center in a protein, enabling direct observation of the unstable intermediates that form following reduction and subsequent reaction steps.37,

38-48

Second, pulse radiolysis does not require

substrates or chemicals; thus, no complications arise from the kinetic constraints imposed by chemical events.37-40, 46, 49 Pulse radiolysis has been used to characterize the quaternary structure of methemoglobin by monitoring the kinetics of O2 binding to the valence hybrid.47, 48 Here, we describe the application of pulse radiolysis techniques to D. radiodurans NOS (DrNOS) for the purpose of monitoring the reaction intermediates essential for substrate oxidation. Intramolecular electron transfer from H4F to heme is essential for suppling the electrons required for O2 activation during sequential NO synthesis. We present the first direct


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observation of the reduction of the ferrous dioxygen-form with H4F within the enzyme on the milliseconds time scale. MATERIALS AND METHODS Protein Expression and Purification. The NOS gene of D. radiodurans (ATCC strain number 13939) was amplified by PCR from genomic DNA. PCR primers harboring an NdeI site before the 5’ start codon and a BamHI site after the 3’ stop codon were used, and PCR reactions were carried out in the presence of 2% dimethyl sulfoxide. The amplified fragment was cloned into a pET15B expression vector. The cDNA constructs were confirmed by DNA sequencing. The pET15b-DrNOS vector was transformed into E. coli BL21 DE3 cells and the cells were grown to saturation. Four milliliters of saturated E. coli BL21 DE3 were inoculated into 400 mL Terrific Broth medium with 50 mg mL-1 ampicillin at 37 °C. After the cultures had reached an optical density of 0.6-0.8 at 600 nm, protein expression was induced by the addition of 1 mM isopropyl β-D-thiogalactopyranoside and 450 µM 5-aminolevulinic acid hydrochloride, followed by incubation over 24 h at room temperature. The cells were then harvested and stored at -80 °C. The DrNOS purification procedure was similar to that reported previously.15, 19 The thawed cells were incubated for 60 min in buffer A (50 mM HEPES, pH 8.0, 300 mM NaCl, 5 mM Arg, 10 mM imidazole, and 10% glycerol) with 0.5 mg mL-1 egg-white lysozyme and 1 mM 4-(2aminoethyl)benzene sulfonyl hydrochloride. The suspension was sonicated on ice, and insoluble debris was removed by centrifugation at 30,000-g for 60 min. The supernatant was applied to a Ni2+-NTA Superflow resin column (Qiagen) that had been equilibrated with buffer A. The column was washed with 200 mL buffer A followed by 200 mL buffer A containing 50 mM imidazole. Proteins were eluted with buffer A containing 200 mM imidazole and were then further purified using size-exclusion chromatography. The DrNOS thus obtained was more than 80 % pure as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The protein


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concentration was determined using an extinction coefficient for ferric-imidazole complex of 97 mM-1cm-1 at 427 nm.19 All other reagents were of the highest grade of purity available and were obtained from commercial sources. Pulse Radiolysis. Pulse radiolysis experiments were performed using a linear accelerator at the Institute of Scientific and Industrial Research at Osaka University.38-46 The pulse width and energy were 8 ns and 27 MeV, respectively. The dose was in the range 15-400 Gy. The sample was placed in a quartz cell with an optical path length of 0.3 - 1 cm, and the temperature of the sample was maintained at 25 °C. The light source for the spectrometer was a 200 W Xe lamp. After passing through an optical path, the transmitted light was analyzed and monitored using a fast spectrophotometric system composed of a Nikon monochromator, an R-928 photomultiplier, and a Unisoku data analysis system. The time-resolved transient absorption spectrum was obtained by focusing the monitor light into a quartz optical fiber that transported the electron pulse-induced transmittance change to a gated spectrometer (Unisoku, TSP-601-02). The temporal resolution of the spectrometer was 10 ns. The samples used in the pulse radiolysis experiments, solutions containing DrNOS (5-50 µM), 0.1 M tert-butyl alcohol (for scavenging OH radicals), 500 µM (Arg or NHA), and 50 µM H4F in 10 mM phosphate buffer (pH 7.4) were deoxygenated in sealed cells by repeated evacuation and flushing with argon. A 0.1 M tert-butyl alcohol concentration had no effect on the enzyme activity or the optical absorption spectrum of NOS. Samples containing O2 or CO were prepared by mixing argon-saturated buffer solutions with the appropriate volumes of O2 or a CO-saturated buffer solution, respectively. The kinetics were analyzed using the following equation: ∆A(t) = α exp(-k1t) + β exp (-k2t)

(1)


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where ∆A is the total intensity change at a certain time, t, after pulse radiolysis, α and β are the initial intensities for each phases, and k1 and k2 are the rate constants. Kinetic Solvent Isotope Effects. Buffer solutions were prepared by lyophilizing phosphate buffers and then dissolving the dry residue in D2O. Stock solutions of DrNOS were mixed with buffer solution (10 mM phosphate buffer, pD 7.4) containing 0.1 M tert-butyl alcohol, H4F, and Arg (or NHA). The final mole fraction of D2O exceeded 90%. Stopped-Flow Analysis. Rapid kinetic measurements were carried out using an RSP-100003DR stopped-flow rapid-scan spectrometer (UNISOKU Co. Ltd., Osaka, Japan), as described previously.50 One chamber of the apparatus contained the ferrous enzyme (5 µM) in 50 mM HEPES buffer (pH 7.4), prepared by adding sodium dithionite (in minimal but sufficient amounts to produce the ferrous form). This solution was transferred into the chamber anaerobically. The other chamber contained the air-saturated buffer. The temperature of the chambers and the sample holder was maintained at 10 °C. Data points were collected every 1 ms during the measurements. The dead time of mixing was of the order of 2 ms. Optical absorption spectra were recorded using a Hitachi U-3000 spectrophotometer.

RESULTS Pulse Radiolysis- Pulse radiolysis experiments involve the instantaneous generation of eaq-, which reduces the heme iron of hemoproteins.32-44 Reduction of the heme iron of DrNOS by eaqwas reflected in the initial increase in the absorbance at 450 nm (Figure 3). Under the conditions employed here, the solutions containing 8 µM DrNOS, 500 µM Arg, 50 µM H4F, 10 mM potassium phosphate buffer (pH 7.4), 30-40 µM O2, and 0.1 M tert-butyl alcohol were irradiated with 80 Gy, which resulted in [eaq-] = 21 µM. The kinetic difference spectrum in the Soret region at 12 µs after pulse radiolysis of DrNOS is shown in the Supporting Information Figure S1. The


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spectrum, which featured broad twin absorption peaks around 420 and 450 nm, was indistinguishable from the spectrum obtained through chemical reduction with sodium dithionite (SI Figure S1). In our reaction systems, eaq- reacted with O2, H4F, and Arg, in addition to the heme iron of DrNOS. The second-order rate constants and the concentrations of each reactants (Table 1) indicated that 2-30 % of eaq- generated could react with the heme in DrNOS. An increase in the absorbance at 450 nm (SI Figure S1) corresponded to 2.5 µM heme reduction, and the yield of the reduction of the heme relative to eaqwas 8 %. This value was similar to that obtained from cytochrome P450cam.35 Although a fraction of eaq- reacted with O2 to form O2-, O2- failed to undergo reactions with DrNOS in the presence or absence of H4F. The addition of Cu/Zn superoxide dismutase (11 µM) had no effect on the reactions (data not shown). In an aerobic solution, the spectral changes associated with the heme reduction were followed via an absorption band composed of two phases: fast and slow phases, with a time-scale on the order of microseconds (Figure 3). In the absence of H4F, the slower phase was absent. The rate of the faster phase increased with the O2 concentration (Figures 4). The Supporting Information Figure S2 compares the kinetic difference spectrum at 100 µs after pulse radiolysis of DrNOS with that initially obtained after mixing O2 with the dithionite-reduced enzyme, as described below. The spectra were essentially identical. These results indicated that the absorbance change in the faster phase corresponded to a bimolecular reaction between O2 and FeII heme of DrNOS. The slope of Figure 4(B) indicates that the second-order rate constant of the reaction was 2.8 × 108 M-1 s-1. It should be noted that the value was approximately 102 times that of neuronal NOS (9 × 105 M-1 s-1), as determined by stopped-flow methods.53 The spectral changes corresponding to the formation of Fe(II)-O2, in the presence of H4F included a slower absorption change at 450 nm on the milliseconds time scale (see the inset of Figure 3). The rate constant of the slower phase in the presence of H4F was calculated to be 2.2 ×


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103 s-1 and was not affected by changing the enzyme and O2 concentrations. (In these experiments, the enzyme concentration was varied between 5 and 20 µM). Therefore, the slower process was attributed to intramolecular electron transfer from H4F to the ferrous-dioxygen intermediate. Figure 5 shows the resulting transient spectrum (red line: 3 ms after pulse radiolysis) and a comparison with the transient spectra of FeII (blue points: 12 µs after pulse radiolysis), FeIIO2 (green points: 100 µs after pulse radiolysis). The spectrum of the intermediate, which featured an absorption maximum at 440 nm and an absorption minimum at 390 nm, was not distinct from that of the ferric form. The intermediate in the absorption spectrum was further converted over a period of seconds, as shown in Figure 6. The absorption band intensity increased and decreased at 400 nm and 450 nm, respectively. The spectroscopic changes observed on the order of seconds were similar to those observed in the single-turnover reaction of ferrous DrNOS during stopped-flow measurements, as shown in Figure S3 and described below. At 3 s, the spectrum was similar to that of the initial oxidized form of DrNOS based on the kinetic difference spectrum after the pulse (Figure 6-B). These results indicated that the absorbance changes in Figure 6-A corresponded to the hydroxylation of Arg by the intermediate of NOS. The first-order rate constant of the reaction at 4 s-1 was calculated. The spectrum recorded after pulse radiolysis indicated that the species returned to the ferric form. The spectroscopic changes and the rate constants associated with the oxidation of NHA were of particular interest. A distinct mechanism involving two reaction steps was proposed (Figure 1).28-31 Similar pulse radiolysis experiments were performed under identical conditions but with 500 µM NHA. Figure 7 shows absorption changes at 450 nm after pulse radiolysis of DrNOS in the presence of NHA and H4F. As in the presence of Arg (Figure 3), the initial rapid increase due to a reduction of heme iron, was followed by fast and slow absorption decrease phases (Figure 7A). In the absence of H4F, the slower phase was absent. The rate constant of the phase observed in the presence of H4F was calculated to be 2.2 × 103 s-1. Figure 7-C shows the transient spectra


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at 12 µs, 100 µs, and 3 ms after pulse radiolysis of DrNOS in the presence of NHA. The spectra were similar to that observed in the presence of Arg. The intermediate observed in the absorption spectra was converted, over a period of seconds, as shown in Figure 7-B, accompanied by an increase in the absorption at 390 nm. The spectroscopic changes in the time domain, on the order of seconds, were similar to those observed in the single-turnover reaction of DrNOS during the stopped-flow experiments; however, the intermediate was found to decay more slowly (0.5 s-1) compared with the intermediate observed in the presence Arg. Kinetic solvent isotope effects have been examined as a useful probe of proton delivery in enzymatic reactions of cytochrome P450.54-57 Experimental evidence for NOS proton and electron transfer events58 was obtained by performing similar pulse radiolysis experiments in D2O buffer. Figure 8 comapres absorption changes after pulse radiolysis of DrNOS in the presence of H4F and Arg or NHA in D2O and H2O. The faster phases observed on the order of milliseconds were not affected significantly by D2O, as shown in Figures 8-A, -B, and Table 2; however, the slower phase in the time domain, on the order of seconds, displayed substantial slowing upon H/D substitution (Figures 8-C and -D). The use of Arg as a substrate provided rate constants of kH2O = 4.0 s-1 and kD2O = 0.38 s-1, providing a 10.5-fold kinetic isotope effect on the reaction rate. By contrast, the rates of NHA were kH2O = 0.5 s-1 and kD2O = 0.2 s-1, providing a 2.5-fold kinetic isotope effect on the reaction rate. Stopped Flow Analysis-Stopped-flow spectroscopy was used to analyze the kinetics of the single-turnover reactions in DrNOS. A dithionite-reduced DrNOS containing Arg and H4F was rapidly mixed with an O2-containing buffer to initiate the reaction. Figure S3 shows the spectral changes observed upon mixing reduced DrNOS with dioxygen in the presence of Arg and H4F. The data were essentially identical to the results reported previously.19 The species observed initially, characterized by an absorption maximum of 420 nm, formed within the dead time of the instrument. These results indicated that the reaction between the ferrous enzyme and O2 was completed within 2 ms. The rapid formation of the FeIIO2 species was shown previously.19 In the


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presence of H4F, the ferrous-dioxygen form was found to decay to the original ferric form of the enzyme on the order of seconds (Figure S3-B), during which time absorption increased and decreased at 400 nm and 450 nm, respectively. A first-order rate constant of 4 s-1 was calculated from both traces. In the absence of H4F, the process became slower (k = 0.1 s-1) (data not shown). The spectra indicated no appreciable presence of the intermediates at any wavelength. Discussion In this study, we observed the following processes after pulse radiolysis of DrNOS (Eq. 2) (Figure 2).

Radiolytically generated eaq- reduced the heme iron of DrNOS with a diffusion-controlled rate.3238

The ferrous enzyme (FeII) thus formed resulted in the rapid formation of a ferrous-dioxygen

complex (FeII-O2) with a second-order rate constant of 2.8 × 108 M-1 s-1. Subsequently FeII-O2 was converted to the intermediate via a slow phase, as shown in Figure 3, only in the presence of H4F. In these processes, the time constants associated with the formations of FeII-O2 and the intermediates were 30 µs (2 → 3) and 320 µs (3 → 4), respectively, as shown in Figure 2. These results suggested that these reactions reached completion within the initial 2 ms in the stoppedflow experiments. The monophasic conversion to the original ferric form which occurred at a rate equivalent to that of Arg-hydroxylation in a single turnover reaction (Eq. 3)(Figure 2), was monitored.59 This model was supported by the pulse radiolysis experiments conducted in the presence of NHA (Figure 7), in which the intermediate decayed at a rate (0.5 s-1) similar to that observed using the stopped-flow method.19 In this study, our results provided the first direct observation of the build-up of a new reaction intermediate involving catalytically active species of Arg or NHA hydroxylation in DrNOS. Additional intermediates involved in the conversion from FeIIO2 to FeIII were observed previously in the context of W188H mutant mammalian


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iNOS26 and Geobacillus stearothermophilus (GsNOS).18 Because the formation of the intermediate in DrNOS (2.2 ×103 s-1) observed here was much faster than that observed (on the

order of seconds), the intermediate was definitely distinct from those observed in the presence of iNOS or GsNOS. The first-order intramolecular electron transfer rate constant associated with the transfer from the bound H4F to FeIIO2 in DrNOS was 2.2 × 103 s -1. This process did not involve any steps other than intramolecular electron transfer and was not affected by the solvent kinetic isotope effect; therefore, the experiments conducted here provided the first direct determination of the electron transfer kinetics. This rate was comparable to the rate of intramolecular electron transfer in nitrite reductase (1.4 × 103 s -1)60, 61 in which the two redox centers were 11-13 Å apart.62, 63 The distance between heme and biopterin across a near van der Waals distance of 3 Å was considered, and the distance between biopterin and FeIIO2 was not thought to exceed 10.3 Å.64, 65 This distance is compatible with intramolecular electron transfer on the submillisecond time scale.66 It is important to study how the NOS proteins tune the redox potentials of biopterin and to understand the factors that promote electron transfer to FeIIO2. Theoretical calculations suggested that intramolecular electron transfer was possible when O2 was doubly protonated.67 It is important to realize that the rate of electron transfer from biopterin to FeIIO2 in DrNOS (2.2 × 103 s -1) in the present work was much faster than the rates measured in mammalian NOS and determined by the rapid freeze-quench EPR (11 s-1).59,

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This process was kinetically

coupled to the decay of FeIIO2 and the rate of Arg hydroxylation (9 s -1). 59 In a mammalian NOS-catalyzed reaction, the reduction of FeII-O2 with pterin appears to be a rate-limiting step for further catalysis.68, 69 This mechanism prevents the accumulation of reactive species downstream of the heme-dioxygen form during substrate hydroxylation. Instead, the monophasic conversion


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of FeIIO2 to the ferric enzyme is observed during a single turnover reaction.68 In the reaction of DrNOS, on the other hand, the rate of Arg hydroxylation (4 s-1) was not kinetically coupled to the decay of FeII-O2 for DrNOS, where the rate-limiting step in DrNOS involved the reaction of the intermediate with the substrate. It should be noted that the rate of the process displayed a solvent kinetic isotope effect (kH/kD = 10.5) (Table 2). These results suggested that proton transfer became rate-limiting in the stages (4) →(5) →(6) of the catalytic process. A similar solvent kinetic isotope effect was observed in the catalytic rates associated with product formation by the Asp251Asn mutant in cytochrome P450cam.70 By contrast, a smaller isotope effect of 2.5 in the second catalytic step suggested that no additional proton transfer was required. The rate of NHA oxidation may have been partially rate-limiting, resulting in a smaller observed isotope effect compared to the first catalytic step. Thus, there may be difference in mechanism between DrNOS and mammalian NOS. However, significant difficulty in comparison of the data obtained for DrNOS and mammalian NOS is that experiments have been done under completely different conditions. Understanding electron transfer and the subsequent processes carried out in the presence of each NOS requires identical conditions. The application of the rapid freezequench EPR method to DrNOS and the pulse radiolysis experiments with various mammalian NOS should be performed. An important issue is the one-electron reduced state of the ferrous-dioxygen-form of DrNOS observed spectrophotometrically. The active intermediate of DrNOS is characterized by an absorption maximum at ~440 nm and minimum at ~390 nm, in the Soret region (Figure 5). As in the cytochrome P450 reactions,71-75 compound I-like species are considered to insert an oxygen atom into the Nω-H bond of Arg;11, 76, 77 however, the absorption spectra of the compound I ferryl derivatives of NOS have not yet been identified. Compound I-like species, including a ferryl π cation radical, usually have a significantly reduced Soret absorbance intensity relative to the intensities obtained from the ferric enzyme.75 The spectrum obtained in our present experiments was distinct from the spectrum obtained from compound I of chloroperoxidase78 or of compound


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I of cytochrome P450.75 On the other hand, cryotrapped reduction intermediates of FeII-O2 cytochrome P450 and GsNOS exhibited an absorption band at ~440 nm.20,

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Recently, the

reduction of a substrate-bound oxygenated form of cytochrome P450cam using pulse radiolysis was shown to form an intermediate with an absorption maximum at 440 nm.37 The species corresponded to a ferric-peroxo species. This is also supported by the solvent kinetic isotope effect in the present study in Figure 8. The large solvent kinetic isotope effect of 10.5 suggested that the rate-limiting step of the substrate oxidation involved proton-assisted O-O bond scission, and is most likely a deuterium exchange of hydropeoxo in the stages (4) →(5) →(6) (Figure 2).80 If so, the observed absorption at 440 nm could be assigned to ferric peroxo (4) or hydroperoxo intermediates (5). Unlike other cytochrome P450-type monooxygenases, no hydroperoxo intermediates (5) could be isolated. Alternatively, we observed one-electron reduction of the FeIIO2 complex to form higher oxidation states of myoglobin, horseradish peroxidase, and cytochrome P450 using the nanoseconds pulse radiolysis experiments at ambient temperatures.38, 39, 49

In these reactions, the spectra did not indicate the formation of ferric peroxo (4), a ferric

hydroperoxo (5) species, at any wavelength. These results suggested that the process associated with converting ferric-peroxo (Fe2+-O2-) to compound I (6) occurred long before 70 ns. The spectrum observed here would, therefore, correspond to the higher oxidation state of DrNOS as the final product. The rate constant associated with the binding of O2 and ferrous heme (2.8 × 108 M-1 s-1) was about 102 times faster than the corresponding rate constants observed for mammalian neuronal and endothelial NOSs (9 × 105 M-1 s-1, 3.4 × 105 M-1 s-1).53, 81 Previous stopped-flow experiments 1conducted by Reece et al19 revealed that O2 binding to ferrous DrNOS was very rapid, and the reaction was reached completion within the instrumental dead time; however, O2 binding kinetics for other bacterial NOS have not yet been determined. Structurally, the catalytic centers of these proteins are nearly identical, with the exception of a Val → Ile substitution adjacent to the heme iron. The conserved valine residue in mammalian NOS, which resides just above the


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heme pocket, is switched to a conserved isoleucine in bacterial NOSs,82,

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83

in which the Ile

residue has been shown to reduce the release rates of the heme-bound NO species.84, 85 Even small motions in the Ile residue may influence the hydrogen bonding network among solvent molecules and heme ligand. On the other hand, O2 binding to various recombinant myoglobins revealed that substitutions in the distal pocket residues resulted in 100-fold changes O2 binding (6 × 105 M-1 s-1 in ValE11Gln yielding 1.2 × 108 M-1s-1 in HisE7Val). The mutations had little effect on CO binding.86 O2 binding appeared to be controlled by the steric or hydrophobic characteristics of the ligand access channel at the heme distal site. The differences in O2 binding may correlate with the apparent Km values of O2 obtained from the various NOSs. The differences among the Km values of O2 in the NOS may reflect the physiological functions of the NOS in various organisms. A low KmO2 (4 µM) may be required for eNOS to increase the blood supply under hypoxic conditions,81, 87 whereas a higher apparent Km O2 (350 µM)88, 89 for nNOS is observed for NOS activity in whole animals and tissues. DrNOS with a high O2 binding constant may indicate an efficient sensing ability at lower O2 concentrations. Finally, the rapid reductions of the heme iron center in DrNOS enabled the first direct determination of the rates of intramolecular electron transfer; however, it is not known whether this process is characteristic of DrNOS. It would be interesting to apply this analysis to other types of NOS, including mammalian NOS and other bacterial NOS. These experiments are being planned in our laboratory.

ASSOCIATED CONTENT AUTHOR INFORMATION Corresponding Author * E-mail: [email protected]. Telephone: +81-6-6879-8501.


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ORCID Kazuo Kobayashi: 0000-0002-5586-9112 Author Contributions Y.T., K.K., and T. K. designed the research. Y. T. and K. K. prepared samples, performed the pulse radiolysis experiments, and analyzed the data. Y. T., F. T., and T. M. conducted stoppedflow experiments. Y. T. and K. K. prepared the manuscript. All authors have given approval to the final version of the manuscript. Funding Sources This work was supported by Grants-in Aid (25246036 to K. T. and K. K.) from the Japanese Ministry of Education, Science and Culture.

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENTS We thank the members of the Radiation Laboratory at the Institute of Scientific and Industrial Research, Osaka University, for assistance in operating the linear accelerator. SUPPORTING INFORMATION AVAILABLE This material is available free of charge via the Internet at http://pubs.acs.org.


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(83) Wang, Z. Q., Wei, C. C., Sharma, M., Pant, K.; Crane, B. R., and Stuehr, D. J. (2004) A Conserved Val to Ile Switch near the Heme Pocket of Animal and Bacterial Nitric-oxide Synthases Helps Determine Their Distinct Catalytic Profiles. J. Biol. Chem. 279, 19018-19025. (84) Beaumont, E., Lambry, J.-C., Wang, Z-Q., Stuehr, D. J., Martin, J-L., and Slama-Schwok, A. (2007) Distal Val346Ile Mutation in Inducible NO Synthase Promotes Substrate-Dependent NO Confinement. Biochemistry, 46, 13533-13540. (85) Whited, C. A., Warren, J. J., Lavoie, K. D., Weinert, E. E., Agapie, T., Winkler, J. R., and Gray, H. B. (2012) Gating NO Release from Nitric Oxide Synthase. J. Am. Chem. Soc. 134, 2730. (86) Springer, B. A., Sligar, S. G., Olson, J. S., and Phillips, Jr. G. N. (1994) Mechanisms of Ligand Recognition in Myoglobin. Chem. Rev. 94, 699-714. (87) Santolini, J., Meada, A. L., and Stuehr, D. J. (2001) Differences in Three Kinetic Parameters Underpin the Unique Catalytic Profiles of Nitric-oxide Synthases I, II, and III. J. Biol. Chem. 276, 48887-48898. (88) Santolini, J., Adak, S., Curran, C. M. L., and Stuehr, D. J. (2001) A Kinetic Simulation Model That Describes Catalysis and Regulation in Nitric-oxide Synthase. J. Biol. Chem. 276, 1233-1243. (89) Abu-Soud, H. M., Wang, J., Rousseau, D. L., Fukuto, J. M., Ignarro, L. J., and Stuehr, D. J. (1995) Neuronal Nitric Oxide Synthase Self-inactivates by Forming a Ferrous-Nitrosyl Complex during Aerobic Catalysis. J. Biol. Chem. 270, 22997-23006.


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Biochemistry

TABLE 1 The rate constants of reaction of eaq- with components of the reaction system of DrNOS Compound

Rate constant (k) (M-1s-1)

Concentration (µM)

O2 Arg H4F NOS

2.2 × 1010 1.53 × 108 2.5 × 1010 3.0 × 1010

20~200 500 50 5~50

Ref

(51) (52) (51) present study


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Table 2 Solvent isotope effects of the rate constants in the faster and slower phases

Substrate

Arg NHA

H2O (s-1)

D2O (s-1)

Kinetic solvent isotope effects

2.2 × 103

1.8 × 103

1.2

4.0

0.38

10.5

3

2.2 × 10 0.5

3

2.0 × 10

1.1

0.2

2.5


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Biochemistry

Figure 1. Reaction catalyzed by nitric oxide synthase.


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Figure 2. Reaction schemes describing the function of the DrNOS enzyme toward Arg hydroxylation. The bracketed heme complexes have not been directly observed during catalysis.


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Biochemistry

Normalized ∆A

Page 33 of 39

+H4F

+H4F 450 nm 0

0

5 10 Time (ms)

450 nm

250 500 Time (µs)

750

Figure 3. Absorbance changes after pulse radiolysis of DrNOS in the presence (red line) or absence (blue line) of 50 µM H4F monitored at 450 nm. Inset: absorbance changes on longer time scales. The reaction mixtures contained 8 µM DrNOS, 500 µM Arg, 10 mM potassium phosphate buffer (pH 7.4), and 0.1 M tert-butyl alcohol.


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Figure 4. (A) O2 concentration dependence of the absorbance changes after pulse radiolysis. (B) O2 concentration dependence on the rate constants observed after pulse radiolysis. Samples contained 8 µM DrNOS and 0.1 M tert-butyl alcohol in a 10 mM phosphate buffer (pH 7.4).


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Biochemistry

Figure 5. Kinetic difference spectra at 12 µs (blue points), 100 µs (green points), and 3 ms (red points) after pulse radiolysis of DrNOS.


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Figure 6. (A) Absorbance changes after pulse radiolysis of DrNOS. (B) Kinetic difference spectraum at 3 s after pulse radiolysis of DrNOS.


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Biochemistry

Figure 7. (A) (B) Absorbance changes after pulse radiolysis of DrNOS, monitored in the presence of 50 µM H4F. The reaction mixtures contained 8 µM DrNOS, 500 µM NHA, 10 mM potassium phosphate buffer (pH 7.4), and 0.1 M tert-butyl alcohol. (C) Kinetic difference spectra at 12 µs (blue points), 100 µs (green points), and 3 ms (red points) after pulse radiolysis of DrNOS. The blue lines represent the curves obtained by doubleexponential fittings (A).


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Figure 8. Solvent kinetic isotope effects of absorbance changes in H2O (red line) and D2O (blue line) after pulse radiolysis of DrNOS in the presence of 500 µM Arg (A), (B) and NHA (C), (D). The reaction mixtures contained 8 µM DrNOS, 50 mM H4F, 10 mM potassium phosphate buffer (pD 7.4), and 0.1 M tert-butyl alcohol. The blue lines represent the curves obtained by doubleexponential fittings (A) (C) and single-exponential fitting (B) (D).


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Biochemistry

TOC graphic specifications

Insert Table of Contents artwork here


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Reaction Intermediates of Nitric Oxide Synthase from Deinococcus

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