Biochimica et Biophysica Acta 1847 (2015) 1240–1244

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Structure and properties of the catalytic site of nitric reductase at ambient temperature

Vangelis Daskalakis a,TakehiroOhtab, Teizo Kitagawa b, Constantinos Varotsis a,⁎ a Department of Environmental Science and Technology, Cyprus University of Technology, P.O. Box 50329, 3603 Lemesos, Cyprus b Picobiology Institute, Graduate School of Life Science, University of Hyogo, Hyogo 678-1297, Japan article info abstract

Article history: reductase (Nor) is the third of the four enzymes of bacterial denitrification responsible for the Received 9 March 2015 − catalytic formation of laughing gas (N2O). Here we report the detection of the (HO–N=N–O ) Received in revised form 13 June 2015 −1 species (νN–N = 1332 cm ) in the heme b3 Fe–FeB dinuclear center of Nor from Paracoccus denitrificans.We Accepted 29 June 2015 have also applied density functional theory (DFT) to characterize the bimetallic-bridging hyponitrite species in Available online 2 July 2015 the reduction of NO to N2O by Nor and compare the present results with those recently reported for the N–N Keywords: bond formation in the ba3 and caa3 oxidoreductases from Thermus thermophilus. Heme proteins © 2015 Elsevier B.V. All rights reserved. Oxidoreductases UV Raman spectroscopy Hyponitrite Density functional theory

1. Introduction atom form the dinuclear, NO reduction site. The molecular mechanism

of the reduction of NO to N2O by Nor and the structures of the key inter- Denitrification is the anaerobic use of bacteria of oxide spe- mediates have not yet been determined. cies as terminal electron acceptors in place of O2. It is the only process The most commonly encountered metals in the biological reduction of that returns large amounts of fixed nitrogen to the atmosphere [1]. NO to N2O are iron and copper in mononuclear, binuclear, and dinuclear The reduction of nitric oxide (NO) to laughing gas (N2O) which is centers. In P450Nor the electrons needed for the reduction of NO + the committed step in denitrification, is also an “escaping” process, are directly transferred from NADH (2NO + NADH + H → N2O+ + resulting in the release of large amounts of N2O, a greenhouse gas that H2O + NAD +H2O) [10]. This way, a single molecule of NO binds at is implicated in atmospheric depletion into the atmosphere the heme Fe, and addition of two electrons to heme Fe3+–NO yields the [1–3]. Several themes have emerged in the studies of biological control two electron reduced species Fe2+–N=O−. A second NO molecule at- of nitric-oxide chemistry [1–5]. Apparently, evolution has clearly select- tacks the N atom of the ferrous–NO species to transiently yield hyponitrite ed means by which to take advantage of the unique NO chemistry, and a (HONNO−), and thus the N–N bond formation. Cleavage of the N–Obond number of enzymes have evolved that are able to activate NO kinetical- produces the ferric enzyme, N2OandH2O. Proposed mechanisms for the ly, and control their redox chemistry. In bacterial respiration the reduc- NO reduction by Nor involve two NO molecules in the dinuclear center. tion of NO to N2O by terminal oxidoreductases supports the hypothesis Based on the Soret and UV–Raman experiments of the reaction of ba3 of a common evolutionary origin of bacterial denitrification and aerobic oxidase with NO, a reaction scheme for the initial binding of two NO respiration. Comparison of the enzymes responsible for the activation of molecules to the oxidized heme a3–CuB binuclearcenterinwhichthefor- NO in denitrification and bacterial respiration may provide the means to mation of the N–N bond required the addition of an electron was demon- identify conserved structural features, which can be assumed to be strated [7,8]. The hyponitrite species is formed subsequent to the 3+ involved in basic functions common to both classes of enzymes [4–12]. formation of a transient heme a3 Fe –NO species and the addition of Nitric oxide reductase (Nor) from Paracoccus denitrificans contains the second NO molecule to CuB. Given that, upon addition of NO to the four redox centers: three heme groups and one non-heme FeB atom oxidized enzyme, heme a3 is in the ferric form, then CuB must be reduced 1+ − [4,5]. Heme c is bound to NorC subunit and functions as the electron upon addition of NO, producing CuB and NO2 . Addition of a second NO 1+ 1+ entry site of the enzyme. NorB contains a low-spin heme b and a five- molecule to CuB yields the CuB –NO complex. coordinate, high-spin heme b3, which, together with a non-heme iron The Soret excitation resonance Raman spectra of the reactions of oxidized and reduced Nor with NO have been reported [11,12].Inthis ⁎ Corresponding author. report we have extended our original work on the NO binding to Nor E-mail address: [email protected] (C. Varotsis). and have applied UV resonance Raman spectroscopy to investigate the

http://dx.doi.org/10.1016/j.bbabio.2015.06.014 0005-2728/© 2015 Elsevier B.V. All rights reserved. V. Daskalakis et al. / Biochimica et Biophysica Acta 1847 (2015) 1240–1244 1241 reaction of NO with the dinuclear center of Nor [11,12]. Our results indi- − cate the formation of the hyponitrite (HO–N=N–O ) species (νN–N = −1 1332 cm )inthehemeb3 Fe–FeB dinuclear center of Nor from P. denitrificans. We have also applied density functional theory (DFT) to characterize the heme Fe/FeB hyponitrite species. With the identifica- tion of the hyponitrite species the mechanism for the 2e−/2H+ reduc- 3+ tion of NO to N2O and the role of the non-heme FeB in Nor versus 2+ CuB in heme-copper oxidases in the reaction mechanism can be described with more certainty.

2. Materials and methods

2.1. Experimental section

Nitric oxide reductase was isolated from P. denitrificans cells accord- ing to previously published procedures [5,11]. The activity of the enzyme was measured according to [5] and was 40 μmol/mg/min (46e-/s). The samples were concentrated to 150 μMin20mMTris pH 7.4 containing 0.05% dodecyl β-D-maltoside and stored in liquid nitrogen until use. The concentration of Nor was determined using 5 −1 −1 an ε411 = 3.11 × 10 M ·cm . Oxidized samples were exposed to 1 atm NO in an anaerobic rotating quartz cell for the Raman measure- ments. The hyponitrite species in Nor was formed upon addition of reductant (final concentration 130 μM NOR/500 μM dithionite) to the NO-exposed oxidized enzyme. The UV Raman spectra were acquired as described previously [8].

2.2. Computational

Fig. 1. UV-resonance Raman spectra of oxidized Nor (trace A), upon addition of 14N16O Hyponitrite complex was studied in cases of singlet through septet (trace B) and 15N16O (trace C) at pH 7.5. Traces D and E are the spectra of the oxidized and doublet through sextet states with +3 and +2 overall charges re- Nor–14N16O and Nor–15N16O complexes, respectively, upon addition of reductant spectively at the riblyp/tzvp level of theory with ecp-10-mdf effective (final concentration 130 μM NOR/500 μM dithionite). Trace B–Trace C is the difference spectrum of the 14N16O-minus 15N16O–Nor adduct. Trace D–Trace E is the difference core potential on iron atoms as implemented in Turbomole 5.8.0 soft- 14 16 15 16 14 16 spectrum of N O- N O. For comparison, the inset shows the ba3- N O minus ware package (Development of the University of Karlsruhe and 15 18 ba3- N O difference spectrum [8]. The excitation laser wavelength was 244 nm and Forschungszentrum Karlsruhe GmbH, 1989–2007, TURBOMOLE the incident laser power 150 μW. Different samples of Nor were accumulated for 4 min GmbH, since 2007; available from http://www.turbomole.com). The each, resulting in a total accumulation time of 100–120 min for each spectrum. quartet species of total charge +2 converged to a structure consistent with our previous DFT study [13] while all other species have either a distorted porphyrin (singlet, doublet), almost one spin (~0.80) localized on porphyrin (quintet, septet), or dissociated hyponitrite substrate the 14NO bound form of the oxidized enzyme (final concentration (octet), and thus, they were rejected. For the sextet species, the vibra- 130 μM Nor/500 μM dithionite). A small but noticeable increase in the tional frequency calculation gave inconsistent results to our previous intensity of the 1339 cm−1 mode is detected. Trace E was obtained 15 DFT study on CuB–Fe binuclear center [13]. after the addition of reductant to the NO-bound form of the oxidized enzyme. A relative decrease in intensity and bandwidth of the 3. Results 1339 cm−1 mode and the appearance of a new mode at 1302 cm−1 is

observed. Similar observations were detected in the reaction of ba3 The Fe3+–NO stretching and the Fe3+–N–O bending modes of ferric with NO, 15NO and 15N18O. The presence of the 1302 cm−1 mode in 15 nitrosyl-heme proteins/enzymes have been detected under Soret both the ba3 and Nor samples upon explosion to NO strongly indicates excitation, [11] however, the N–O stretching mode in the 1900– the formation of the same species in both enzymes. The difference spec- 1925 cm−1 region has been observed only under UV-excitation [14].It trum D–E(14N16O–15N16O) show the presence of a weak peak/trough −1 was suggested that there is little orbital conjugation between the NO pattern at 1332/1300 cm , as compared to that observed in ba3 ligand and the heme and because such conjugation is not necessary to (Fig. 1,inset)[8]. We suggest that the weak intensity of the peak/trough occur for UV–RR detection, it was proposed that the UV–RR enhance- pattern at 1332/1300 cm−1 is due to the low concentration and/or short ment of the N–O stretching mode is the result of a localized π–π⁎ tran- life time of the observed Nor/NO species. In the sodium, silver and mer- sition [14]. RR excitation in the UV region enhances strong bands cury salts of the hyponitrous acid the fundamental frequency of the N–N derived from aromatic residues such as tyrosine (Y) and tryptophan bond of the hyponitrite ion (HO–N=N–O−)isat1392cm−1 [15].We (W) [14]. assign the mode at 1332 cm−1 in Nor as the N–N stretching vibration of Fig. 1 (trace A) shows the UV–RR spectrum of oxidized Nor (trace the hyponitrite species because of the 15N16O isotope shift and the sim-

A) and the NO bound forms at pH 7.5 in the absence (traces B and ilarity in the frequency with that observed in ba3, and the similarity of C) and presence (traces D and E) of reductant. The strong bands in the its frequency to that reported for the hyponitrite ion (HO–N=N–O−) spectra arise from 1618 (Y8a, W1), 1553 (W3), 1458 (W5), 1359 [8,15].The6cm−1 difference between the hyponitrite-bound to the 14 16 15 16 (W7) and 1339 (W7). Traces B ( N O) and C ( N O) are of the binuclear center of ba3 and that bound in the dinuclear center of Nor oxidized NO-bound form (heme Fe3+–NO). The difference spectrum indicates that the N–N bond has less double bond character when B–C shows that the formation of the heme Fe3+–NO complex is not bound to Nor. The small intensity of the 1332/1300 cm−1 pair, as −1 followed by protein changes that could be detected in the spectrum. compared with that observed at 1338/1300 cm in ba3, indicates the Trace D was obtained after addition of four equivalents of reductant to small population of the hyponitrite species detected under our 1242 V. Daskalakis et al. / Biochimica et Biophysica Acta 1847 (2015) 1240–1244

experimental conditions. Our assignment is further supported by the similarity in the frequency, and the nitrogen and oxygen isotope shifts to those predicted by density functional theory (DFT) calculations [13, 16]. To assign the experimental vibrational peaks, we have performed vi- brational frequency calculations at the b3lyp/6–31 g* and blyp/tzvp levels of theory for the dinuclear hyponitrite model of Fig. 2, as well as for a simpler hyponitrite species (HO–N=N–OH), where two protons substitute for the two iron metals. In the model of Fig. 2 the glutamate

ligand of FeB is not included. The ν(N–N) was calculated to be 1305 and 1164 cm−1,fortheb3lyp/6–31 g* and blyp/tzvp levels of theory re- spectively, without scaling, for this latter simpler model. Kuhn et al. [15]

measured Raman spectra of Na2N2O2 aquatic solution and assigned the frequency at 1392 cm−1 to the ν(N–N) stretching vibration. Different scaling factors could be derived for the two levels of theory used above and especially the ν(N–N): 1.067 (b3lyp/6–31 g*) and 1.196 (blyp/tzvp) based on the experimental result of Kuhn et al. [15] that can be directly associated with the smaller theoretical model we have

employed. Therefore, the experimental stretching mode ν5(N–N) for the hyponitrite intermediate in the dinuclear center of Nor, modeled blyp/tzvp −1 in Fig. 2, is expected around ν5(N–N) × 1.196 = 1353 cm ,if we apply the scaling factor for the blyp/tzvp level of theory to the

ν5(N–N) theoretically calculated vibrational frequency (see Fig. 3 right and Table 1). The calculated vibrational frequencies for the hyponitrite species in the dinuclear center of Nor along with the calculated isotopic shifts are shown in Table 1 (without scaling). Respective eigenvectors for the normal modes (14N16O) are shown in Fig. 3. The results at both levels of theory appear in Fig. 3, with the b3lyp/6–31 g* level of theory Fig. 2. Geometry optimized structure of the hyponitrite species at the Density Functional in parenthesis. Theory (DFT) level. Mulliken spin populations, along with partial charges in parenthesis For the optimized structure of the hyponitrite complex (quartet, are shown in the inset. Selected interatomic distances are also depicted for comparison. total charge of +2) shown in Fig. 2, almost one spin (0.93) is located

Fig. 3. Normal modes for a small hyponitrite model (left) and the hyponitrite species (right). The theoretical vibrational frequencies at the blyp/tzvp and b3lyp/6–31 g* levels of theory are blyp/tzvp blyp/tzvp −1 shown; the latter appear in parenthesis. We note that a scaling factor can be introduced to ν5(N–N) as ν5(N–N) × 1.196 = 1353 cm (see text). V. Daskalakis et al. / Biochimica et Biophysica Acta 1847 (2015) 1240–1244 1243

2+ Table 1 [8,18]. In the ba3 and caa3 from T. thermophilus the ability of CuB to Calculated vibrational frequencies of hyponitrite and porphyrin modes at the blyp/tzvp − produce NO2 upon reaction with NO and the common reaction mecha- level of theory without a scaling factor, depicting isotopic shifts. The frequencies for the nism for the formation of the hyponitrite species in the heme a Fe/Cu hyponitrite species are shown in bold. We note that a scaling factor can be introduced to 3 B blyp/tzvp blyp/tzvp −1 binuclear centers of both oxidoreductases indicates unique evolution ν5(N–N) as ν5(N–N) ×1.196=1353cm (see text). pathways in respiration. Our data offer direct evidence for the formation 14 16 15 16 14 18 15 18 Hyponitrite modes N O N O N O N O of the hyponitrite intermediate and provide the isolated marker mode ν (Ν = Ο) 1361 1351 1353 1321 −1 6 of νN=Nat 1332 cm which should be useful for kinetic experiments. ν5 (Ν–Ν) 1131 1105 1129 1102

ν4 (Ν–ΟΗ) 914 886 896 873

ν3 665 659 641 634 5. Conclusions

ν2 541 535 540 529 ν 1 235 235 233 231 In summary, we have applied UV–Raman spectroscopy to probe the 14Ν16Ο 15Ν16Ο 14Ν18Ο 15Ν18Ο Porphyrin modes formation of the hyponitrite (HO–N=N–O−) species from two NO ν 1540 1541 1541 1541 10 molecules in the heme b Fe–Fe dinuclear center of nitric oxide reduc- ν2 1504 1503 1503 1503 3 B fi ν ν3 1421 1422 1422 1422 tase (Nor) from P. denitri cans under steady state conditions ( N–N = ν −1 4 1317 1316 1317 1321 1332 cm ). We have focused our DFT calculations mainly on theoret- ical vibrational spectroscopy on the active site only and not on energet- ics of the mechanism of action [19]. Therefore, no conclusions can be 3+ on heme-iron, indicating a ferric (Fe ). The calculated drawn regarding the actual structure of the detected hyponitirite inter- spin population (2.18) of non-heme FeB, with coordination number 4, mediate in the active site of Nor. Quantum Mechanical/molecular me- indicates a ferrous at the triplet intermediate-spin (IS) state. This way, chanical (QM/MM) simulations will be applied to probe the dynamics the ground-state electronic structure of this complex is described as of the heme Fe–FeB center and the protonation events in the biochemi- having two iron centers of low and intermediate-spin coupled across cal reduction of NO to N2O, in future work. the bridge. It should be noted that in the presence of glutamate which is coordinated to the non-heme iron (absent in our structure) the Transparency document non-heme iron is high-spin [17]. Iron dinuclear center in hyponitrite … converged to a structure with a Fe Fe separation of 4.73 Å and contains The Transparency document associated with this article can be – a dimerized NO [17] exerting a N N distance of 1.31 Å, which is consis- found, in the online version. tent with our previous DFT study on the Cu–Fe hyponitrite complex [13] and assumes an intermediate spin state for the non-heme iron. There Acknowledgments are 6 normal modes 3 × 4–6 = 6 related to the bridged hyponitrite sub- strate ligand. ν through ν vibrational frequencies were also calculated 1 6 This work was supported by funds from the Cyprus Research for the 15N16O, 14N18O and 15N18O substrates to count for the isotopic Promotion Foundation to C.V. (TEXNOLOGIA/THEPIS/0609(ΒΕ)/05), shifts. Results are shown in Table 1. Respective eigenvectors for the and Science, Sports and Culture, Japan (T.K. 14001004). T.O. thanks normal modes (14N16O) are shown in Fig. 3. JSPS for a research fellowship. The DFT calculations were performed in The reason for the two levels of theory is to compare the results of the Univ. of Crete. We thank Prof. George Frudakis for the access to the hyponitrite species in the Fe–Fe center with those in the Fe–CuΒ B the computational facilities. center at the b3lyp/6–31 g* level of theory [13], and also to compare the results between different levels of theory that show to converge. 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