Direct electron transfer from pseudoazurin to nitrous oxide reductase in catalytic N2O reduction

Journal of Inorganic Biochemistry 115 (2012) 163–173

Contents lists available at SciVerse ScienceDirect

Journal of Inorganic Biochemistry journal homepage: www.elsevier.com/locate/jinorgbio

Direct electron transfer from pseudoazurin to nitrous oxide reductase in catalytic N2O reduction Koyu Fujita a,⁎, Mika Hirasawa-Fujita b, Doreen E. Brown a, Yuji Obara b, Fumihiro Ijima a, Takamitsu Kohzuma b, David M. Dooley c a b c

Department of Chemistry and Biochemistry, Montana State University, Bozeman, MT 59717, USA Institute of Applied Beam Science, Ibaraki University, Mito, Ibaraki 310-8512, Japan Office of the President, Green Hall, 35 Campus Avenue, University of Rhode Island, Kingston, RI 02881, USA

a r t i c l e

i n f o

Available online 26 July 2012 Keywords: Electron transfer Copper protein Protein interactions Electrochemistry

a b s t r a c t Pseudoazurin (PAz), a well-characterized blue copper electron-transfer protein, is shown herein to be capable of mediating electron transfer to the nitrous oxide reductase (N2OR) from Achromobacter cycloclastes (Ac). Spectroscopic measurements demonstrate that reduced PAz is efficiently re-oxidized by a catalytic amount of N2OR in the presence of N2O. Fits of the kinetics resulted in KM (N2O) and kcat values of 19.1±3.8 μM and 89.3± 4.2 s−1 respectively. The KM (PAz) was 28.8±6.6 μM. The electrochemistry of Ac pseudoazurin (AcPAz) in the presence of Ac nitrous oxide reductase (AcN2OR) and N2O displayed an enhanced cathodic sigmoidal current–potential curve, in excellent agreement with the re-oxidation of reduced AcPAz during the catalytic reduction of N2O by AcN2OR. Modeling the structure of the AcPAz–AcN2OR electron transfer complex indicates that AcPAz binds near CuA in AcN2OR, with parameters consistent with the formation of a transient, weakly-bound complex. Multiple, potentially efficient electron-transfer pathways between the blue-copper center in AcPAz and CuA were also identified. Collectively, the data establish that PAz is capable of donating electrons to N2OR in N2O reduction and is a strong candidate for the physiological electron donor to N2OR in Ac. © 2012 Elsevier Inc. All rights reserved.

1. Introduction Nitrous oxide reductase (N2OR, EC 1.7.99.6) catalyzes the two-electron reduction of N2O to N2 and H2O in the final step of the denitrification pathway as shown below [1,2]. −

−

NO3 →NO2 →NO→N2 O→N2 The final step in denitrification has important environmental implications. N2O is highly stable in the atmosphere and its greenhouse gas effect is about 300-fold more potent than that of CO2 [3–5], suggesting its contribution to global warming may be significant. N2O may also contribute to ozone depletion [6,7]. Hence understanding the mechanism and regulation of N2O reduction by N2OR, including the critical electron transfer (ET) steps involved, may have broad implications. N2OR is comprised of two identical subunits each containing two copper clusters identified as CuA and CuZ [8–11]. The dinuclear CuA center is the primary electron acceptor from physiological electron donors and also transfers electrons to the catalytic CuZ center [12,13]. The CuZ center is a unique μ4-sulfur bridged tetranuclear site, supported by seven histidines, where N2O binding and reduction ⁎ Corresponding author at: Department of Anesthesiology, University of Michigan Medical School and Veterans Affairs Medical Center, Ann Arbor, MI 48105, USA. E-mail address: [email protected] (K. Fujita). 0162-0134/$ – see front matter © 2012 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.jinorgbio.2012.07.013

take place [9,14,15]. The latest crystallographic results for N2OR from Pseudomonas stutzeri (Ps) comprise a very important and unexpected milestone in our understanding of the tetranuclear CuZ center: the discovery of the additional inorganic sulfur bridge [16]. We believe the new crystallographic data and the results reported herein are entirely consistent. Although substantial experimental evidence has been presented that the fully reduced [4Cu(I)] form of CuZ is the catalytically competent state of N2OR [17–20] others have presented plausible arguments for other Cuz states to fulfill this role [21] . Pseudoazurin (PAz) and c-type cytochromes have been identified as possible physiological electron donors to the denitrification pathway based on computational simulations, docking studies and general spectroscopic and mechanistic investigations [22–25]. For example, PAz performs as an electron donor to cytochrome c peroxidase [26] and N2OR from Paracoccus pantotrophus (Pp) in vitro [27] and to the copper containing nitrite reductase (NiR) that catalyzes the second step of denitrification [28,29]. Recently, Dell'Acqua et al. reported that cytochrome c552 (cytc552) is a physiological electron donor to N2OR from Marinobacter hydrocarbonclasticus (Mh, renamed from Pseudomonas nautica) based on kinetics, NMR and computational docking studies [30]. In Mh, cytc552 is also believed to be an electron donor to cytochrome cd1, the alternative heme-containing NiR [31]. Several c-type cytochromes have been identified in Mh [32,33], but not pseudoazurin. The methylotrophic organism Hyphomicrobium denitrificans, contains

164

K. Fujita et al. / Journal of Inorganic Biochemistry 115 (2012) 163–173

both a blue copper protein similar to PAz [34] and a cytochrome c550 [35]. Although the physiological donor to its N2OR has not been established, it appears that the cytochrome may transfer electrons to the copper-containing NiR [35,36]. In this paper we present compelling evidence that Achromobacter cycloclastes pseudoazurin (AcPAz) is the physiological electron donor to A. cycloclastes N2OR (AcN2OR), using spectroscopic, kinetics and electrochemical approaches. We show that reduced AcPAz is rapidly oxidized by reductively activated AcN2OR in the presence of N2O, and that an enhanced direct current is apparent in the cyclic voltammogram of AcPAz under catalytic conditions. The probable protein–protein interface in the electron-transfer complex is defined and electron-transfer pathways to the CuA site in AcN2OR are identified. In addition, we demonstrated that cytochrome c555 from Ac (Accytc555), does not donate electrons to AcN2OR. Other evidence in support of the role of AcPAz in electron transfer is also presented. 2. Experimental procedure 2.1. Materials Bovine heart cytochrome c (BHcytc), N-cyclohexyl-2-aminoethanesulfonic acid (CHES), cyanogen bromide activated-Sepharose 4 Fast Flow resin, DNAse and ethanolamine were purchased from Sigma-Aldrich. 4,4-dithiodipyridine (4-pyds) was purchased from Acros Organics. All other buffers and reagents were purchased from Fisher Scientific and used without further purification. 2.2. Protein purification As-purified AcN2OR was purified from Ac/pMLnos, as described previously [17] and under anoxic conditions. Oxidized AcN2OR ([2Cu(I)·2Cu(II)] in CuZ and [Cu(I)·Cu(II)] in CuA) was obtained by the treatment of 800 μL of 304 μM as-purified AcN2OR with 100 eq. of K3[Fe(CN)6] followed by the removal of excess K3[Fe(CN)6] via a PD-10 in an anoxic chamber (2% H2 in N2 atmosphere, Coy Labs). AcPAz was expressed and purified as reported earlier [37] with the following modifications: Harvested cells were suspended in 20 mM phosphate, pH 6.0, containing a small amount of DNAse and sonicated. After centrifugation, a 40% ammonium sulfate precipitation was performed and the supernatant was dialyzed versus 20 mM phosphate, pH 6.0 overnight. The solution was applied to a CM-Sephadex column (Bio-Rad, 13 cm × 2.5 cm diameter) and washed with 50 mM phosphate, pH 6.0. The protein was eluted using a gradient of 50 mM to 110 mM phosphate, pH 6.0. A. cycloclastes cytochrome c555 (Accytc555) was isolated from Ac/pMLnos grown at pH ~ 8.0. The purification protocol is detailed in the Supplementary information. The purity of AcN2OR, AcPAz and Accytc555 was confirmed by SDS-PAGE. Protein concentrations were determined using the BCA method [38], the absorbance at 280 nm or the absorbance at 594 nm (oxidized PAz, ε = 3.7 mM −1 cm −1) [39]. Concentrations of AcN2OR are reported as the monomer form.

reaction was initiated by the addition of N2O into the cuvette and monitored using a Cary 6000i spectrophotometer equipped with a thermo-controller (Varian). N2O concentrations were estimated as previously described [17]. The values of KM (N2O) and kcat were determined from the direct fits of the Michaelis–Menten equation (Origin 7.0, Microcal). The KM (PAz and BV) was determined as a function of AcPAz and reduced benzyl viologen (BV) concentrations in the presence of saturated N2O (702 ± 42 μM). Reduced BHcytc and Accytc555 were obtained under the same conditions as reduced AcPAz described above. In the kinetics assay, the reaction was initiated by the addition of N2O into the solution containing 32.7 μM BHcytc or 10.7 μM Accytc555 and 4.9 nM AcN2OR (Supplemental information). 2.4. Electrochemical measurements of AcN2OR and AcPAz Electrochemical measurements were carried out using a model 610C electrochemical analyzer (ALS) or a CV50W (BASi) in 100 mM phosphate at 25 °C. A 4-pyds/Au, an Ag/AgCl electrode and a platinum wire were used as working, reference, and counter electrodes respectively. The 4-pyds/Au electrode was prepared as follows: a gold electrode with a diameter of 1.6 mm was polished on a Texmet/alumina polishing pad (BASi) with a polishing alumina emulsion (0.05 μm, BASi). Subsequently the electrode was sonicated for 5 min and rinsed with double-deionized H2O followed by an immersion into a filtered aqueous saturated 4-pyds solution for 30 min, and an additional rinse with H2O. O2 was removed from the working compartment by passing N2 gas through the electrochemical cell for 30 min. The cyclic voltammograms were recorded at a scan rate of 2.0 mV/s and potentials were converted to these based on a normal hydrogen electrode. We determined that the catalytic current was dependent on the scan rate over 5.0 mV/s. The ET reaction between AcPAz and as-purified AcN2OR was investigated in the absence and presence of N2O. N2O gas was introduced into 1 mL of an AcPAz-containing solution for 20 min and a catalytic amount (5–20 μL) of AcN2OR was subsequently added. The final concentrations of AcN2OR and AcPAz were 0.5–4 μM and 100 μM, respectively. MV-activated AcN2OR was carried out as previously described [19] using the anoxic chamber. The AcPAz-containing solution was purged with N2O gas purified through 3% pyrogallol in a 12 M NaOH solution. Electrochemical measurements were performed in an Ar-filled glove bag. Accytc555 and control measurements using BHcytc were performed similarly. The ET rate constants between AcPAz and AcN2OR were determined using Eq. (a), at various pH conditions between 5.5 and 9.0. 1=2

i ¼ nFACPAz ðkET DPAz CN2OR Þ

······

ðaÞ

CPAz, CN2OR and DPAz are the concentrations of AcPAz and AcN2OR in mol·cm−3 and the diffusion coefficient of AcPAz (2.32× 10−6 cm2 s−1) [39] respectively. kET is the ET rate constant in M−1 s−1, A is the surface area of the electrode in cm2, i is the current in amperes, and F, the Faraday constant, has its usual significance [40].

2.3. Kinetics assay of AcN2OR using AcPAz 2.5. Docking simulation The following steps were performed in the exclusion of dioxygen. “As-purified” AcN2OR was activated using reduced methyl viologen (MV) as reported previously [17,19]. This activation step produces the catalytically relevant 4Cu(I) state in CuZ which was previously identified as the state that reacts directly with substrate [19]. AcPAz was reduced with an excess amount of 100 mM sodium dithionite (DT). The excess DT was subsequently removed using a Superdex 200 column (GE Healthcare, 62 cm × 1.6 cm diameter) equilibrated with 50 mM phosphate at pH 7.1. For the assay, 2 μL of activated AcN2OR (7.5 μM) was mixed with reduced AcPAz (137.8 ± 6.7 μM, TV = 3 mL). The final concentration of AcN2OR was 4.9 nM. The

Docking computations were carried out using the algorithm BiGGER developed by Palma et al. [41]. The structural sources of AcN2OR (2IWF), reduced AcPAz (1BQR), oxidized AcPAz (1BQK), BHcytc (2B4Z), MhN2OR (1QNI) and cytc552 (1CNO) were obtained from the RCS Protein Data Bank (PDB). After all heteroatoms and water molecules were removed, hydrogen atoms were added to the structural information and minimized using the force field parameter Amber94, via Tinker 4.2 software [42]. The minimized coordinates were re-converted to PDB format and the prosthetic groups (Cu centers and the heme groups) were added back to the coordinates. The

K. Fujita et al. / Journal of Inorganic Biochemistry 115 (2012) 163–173

algorithm was performed on the target protein AcN2OR with both AcPAz and BHcytc as probes. For comparison, a docking simulation was also performed on the target protein MhN2OR with cytc552. We generated 5000 candidates for each complex based on the criteria identified in Ref. [41] and ranked them according to a global score and hydrophobic score. The top 20 candidates for each of the four complexes were identified after an additional filter (distance between the CuA center and the redox center for PAz or the CBC portion of the heme group for cytc (b20 Å)) was applied. The 20 candidates were then individually evaluated by the protein–protein interface analysis server, PROTORP [43] to calculate the ΔASA of the complex, the gap volume index, and planarity [43–45]. To determine a plausible ET pathway, we followed the protocol described in Matilla and Haltia [25] using the PATHWAYS algorithm [46,47] implemented in the Harlem molecular modeling program [48]. 2.6. Reduction of oxidized AcN2OR using reduced AcPAz Reduced PAz with no residual dithionate (20 μL aliquots of 1.29 mM stock concentration, up to 20 equivalents) was titrated into oxidized AcN2OR (120 μM initial concentration) in an anoxic cuvette. The reaction mixture was loaded on to either a Superdex 200 column or a Sephadex G50 (Pharmacia, 60 cm × 2.5 cm diameter) that was equilibrated with 50 mM phosphate (pH 7.1) under anoxic conditions. AcN2OR was separated from excess AcPAz under anoxic conditions. Isolated AcN2OR was concentrated down to 74 μM using either an Amicon device with a YM30 membrane or a spin concentrator and loaded into an EPR sample tube. After the concentrations of AcN2OR were determined based on A280, EPR spectra of the samples were collected at 8–30 K using a Bruker EMX-100 spectrometer equipped with a cryostat cavity and a liquid helium transfer line. EPR spectra of 25–100 μM CuSO4 in 100 mM triethanolamine were also collected as a standard for the following spin quantifications. Spin quantifications of the samples were calculated based on the standard plot of the double integration of the EPR spectra as a function of [CuSO4]. Double integrations of EPR spectra were processed using Origin 7.0. 2.7. Quantitative Western blot Wild-type Ac and Ac/pMLnos were lysed using CelLytic B (Sigma), in the presence of DNAse I (Sigma) and protease inhibitor cocktails (Roche). After centrifugation, the supernatants (1 μL of an 8 × dilution for PAz-detection and 1 μL of 200 × dilution for N2OR detection) and standard pure protein samples (PAz: 11–355 ng, N2OR: 4–114 ng) were loaded and separated on 12.5% polyacrylamide gels. The resolved proteins were transferred onto a polyvinylidene difluoride membrane (Millipore) using a Criterion Blotter (Bio-Rad) containing transfer buffer (25 mM Tris and 192 mM glycine) and 20% methanol. The membrane was soaked in 2% bovine serum albumin (BSA)containing TBS (137 mM NaCl and 25 mM Tris at pH 8.0) buffer with 3 mM KCl for 16 h at 4 °C. Blots were incubated with either a rabbit anti-NosZ antibody or a rabbit anti-PAz antibody for 3 h at 4 °C, washed 4 times with rinse buffer (15 mM NaCl, 10 mM Tris and 0.05% Tween 20) and then further incubated with goat antirabbit IgG antibody conjugated to alkaline phosphatase (Zymed) for 1 h at 4 °C. Blots were washed again (4×) with rinse buffer and finally incubated in a premixed 5-bromo-4-chloro-3-indolyl phosphate (BCIP)/nitro-blue tetrazolium (NBT) solution (Sigma) for 10 min (PAz) and 4 min (N2OR) respectively. Immunoblots were scanned and densitometric analyses of the bands were measured using the ImageJ (v.1.57) program from NIH [49]. The contrast intensity from the densitometry measurements were plotted as a function of concentrations corresponding to purified AcPAz and AcN2OR used in the immunoblots (ranges were 0–178 ng for AcPAz and 0–57 ng for AcN2OR). The

165

physiological concentrations of AcPAz and AcN2OR from Ac/pMLnos and wild-type Ac were extrapolated from the regression equations (Origin, Microcal). All data were run in triplicates. 3. Results 3.1. Re-oxidation of reduced AcPAz in catalytic N2O turnover The reduction of one N2O molecule consumes two electrons corresponding to the oxidation of two reduced AcPAz molecules. Fig. 1A shows the re-oxidation of reduced AcPAz in the presence of a catalytic amount of MV-activated AcN2OR after the addition of N2O. The increase in absorbance at 594 nm, which is λmax for oxidized PAz [37], is an unambiguous indication that the ET reaction from reduced AcPAz to AcN2OR occurs. The initial rate of re-oxidation is dependent upon the concentrations of both N2O (Fig 1B) and AcPAz (Fig. 1C). Based on the Michaelis–Menten fits, the KM (N2O) and kcat are 19.1 ± 3.8 μM and 89.3 ± 4.2 s −1 respectively. The KM (AcPAz) is 28.8 ± 6.6 μM. In the presence of a large amount of AcPAz (1.13 mM), the initial rate was decreased to 59.9 ± 3.9 s −1, indicative of substrate inhibition. The Michaelis constant (N2O) is comparable to the non-physiological donor, benzyl viologen (BV, 24.9 ± 3.7 μM) [17] however, the turnover number reported for BV is nearly 2× greater (162.9 ± 4.2 s −1) [19]. The kinetics data are summarized in Table 1. In contrast to the spectral changes observed with the addition of AcPAz to AcN2OR, no absorbance changes associated with reoxidation were observed when reduced bovine heart cytochrome c (BHcytc) or the potential physiologically relevant cytochrome c, Accytc555 was added to MV-activated AcN2OR despite the fact that the redox potential of PAz (260 mV versus NHE, normal hydrogen electrode) is nearly identical with that of BHcytc [39]. Further, while we have not determined the redox potential for Accytc555, we assume the value to be similar to that reported for the cytochrome c554 in Ac (190 mV, pH 7.5) due to its similar spectral properties and chromatographic behavior [50]. 3.2. Direct catalytic electrochemical current via electron transfer between AcN2OR and AcPAz The current–potential curve of AcPAz showed a well-defined quasi-reversible ET process. The electrochemical behavior of AcPAz was unchanged in the presence of a catalytic amount of as-purified AcN2OR. However, once N2O was injected into a solution of AcPAz with AcN2OR, the cyclic voltammogram was drastically altered so that the cathodic sigmoidal current–potential curve was enhanced (Fig. 2 bold line). The large sigmoidal electrochemical response reflects the appearance of the catalytic current, indicative of the regeneration of oxidized AcPAz by AcN2OR. Because the system behaves as a twocomponent catalytic reaction cycle at saturating substrate, the existing theory for the steady-state ECcat, (irreversible chemical catalytic reaction coupled with ET at the electrode) can be applied [40,51]. It was surprising that we did not observe a substantial catalytic current in the experiments using PAz and MV-activated AcN2OR, (Fig. 2-solid line). We attribute this behavior to the inhibitory effect of residual reduced MV from activated AcN2OR (vide infra) in the electrochemical assay. Fig. 3A depicts the catalytic current (nA) as a function of pH with a maximum value near pH 6. Because the redox potential of AcPAz does not change between pH 6.0 and 9.0 [39], the electrochemical measurements were performed under these pH conditions. The catalytic current of AcPAz–AcN2OR is also dependent on the concentration of AcN2OR (Fig. 3B). The plot of the catalytic current as a function of the square-root of [AcN2OR] shows a linear relationship (Fig. 3C) in support of the ECcat mechanism [40]. The second-order intermolecular ET rate constant (kET) was calculated at (3.98± 0.04) × 10 5 M−1 s−1 based on Eq. (a). This kET value is comparable to those reported from AcPAz–

166

K. Fujita et al. / Journal of Inorganic Biochemistry 115 (2012) 163–173 Table 1 Kinetics parameters of AcN2OR and MhN2OR. Donor

AcN2OR

AcPAz BV

MhN2OR

b

Cytc552 MV

kcat (s−1)

89.3 ± 4.2 162.9 ± 4.2 3.8 ± 1.3 321 ± 27

a

KM (N2O) (μM)

Vmax (U/mg)

KM (donor) (μM)

Vmax (U/mg)

19.1 ± 3.8

82.4 ± 3.9 124.0 ± 3.5a NA 128 ± 17

28.8 ± 6.6

113.9 ± 13.5

13.8 ± 2.8

165.6 ± 8.6

50.2 ± 9.0 11.5 ± 3.6

1.8 ± 0.6 157 ± 13

24.9 ± 3.7a NA 14.0 ± 2.9

[AcPAz] = 138 μM and [reduced BV] = 143 μM for 4.9 nM activated AcN2OR, [cytc552] = 7.5 μM and [reduced MV] = 91 μM for 35 nM activated MhN2OR. [N2O] = 702 μM for 4.9 nM activated AcN2OR and [N2O] = 1.25 mM for 70 nM activated MhN2OR. U = μmoldonormin−1. a See Ref. [17]. b Ref. [30].

observed using MV-activated AcN2OR and a relatively smaller amount of reduced AcPAz (AcPAz:AcN2OR = 200:1) in the solution assay. When activity was measured under conditions similar to those in the electrochemistry experiments (AcPAz:AcN2OR = 100:1), AcN2OR activity was substantially reduced (12.9 U/mg, Table 2). A higher activity (77.4 U/mg) was observed only when a large excess of AcPAz over AcN2OR (26,000:1) was present in the assay (Table 2). At these conditions, AcPAz appears to mitigate the inhibitory effect of residual reduced MV thereby enabling catalytic turnover. Finally, the electrochemical behaviors of both BHcytc and the potential physiological donor Accytc555–AcN2OR were also tested in the presence of N2O. In each case, we did not observe an enhanced cathodic current–potential curve. These results are consistent with no re-oxidation of the reduced cytochromes by activated AcN2OR under catalytic conditions.

3.3. Docking simulation between AcN2OR and AcPAz

Fig. 1. Re-oxidation of reduced AcPAz in catalytic N2O turnover. A, Re-oxidation of PAz (152 μM) by MV-activated AcN2OR (4.9 nM) in the presence of saturated N2O. B, Steady-state kinetics of MV-activated N2OR (4.9 nM) mediated by reduced PAz (138 μM) with respect to N2O concentrations. Rates were calculated based on the growth at 596 nm as a function of time. The solid line represents catalysis by reduced PAz. The broken line represents the catalysis by reduced BV for comparison to that by PAz [17]. C, Steady-state kinetics of MV-activated AcN2OR (4.9 nM) with respect to PAz concentrations in the presence of N2O (702 μM). The solid line is a fit of the data to a substrate inhibitory equation, V = Vmax[PAz] /{KM + [PAz](1 + [PAz] /Ki)}, yielding 104Vmax = 19.0 ± 2.3 μmolPAzsec−1, KM = 28.8 ± 6.6 μM and Ki = 0.59 ± 0.30 mM.

AcNiR (6.66× 105 M−1 s −1 at pH 6.0) [29] and from cytc552–MhN2OR (5.60× 105 M−1 s−1 at pH 7.0) [52]. Incubation of AcPAz and as-purified AcN2OR in the presence of reduced MV dramatically reduced the catalytic current (Fig. 4A., Table 2). The inhibition was time dependent and calculated at k = 0.23 min −1, (Fig. 4B). This result is in good agreement with the value reported by Dell'Acqua's et al. (k = 0.30 min −1) for cytochrome c552 and MV-activated MhN2OR [52]. These observations establish that the presence of reduced MV inhibits electron transfer from PAz to N2OR in the electrochemistry experiments. Interestingly, the inhibitory effect of MV may also account for the decreased specific activity

In order to probe the molecular basis for the apparent selectivity for electron-transfer between AcPAz and AcN2OR, 5000 candidates were generated for each of four complexes analyzed using the BiGGER algorithm [41]. The four complexes were: reduced AcPAz– AcN2OR; oxidized AcPAz–AcN2OR; BHcytc–AcN2OR; and cytc552– MhN2OR. We found no significant differences in the docking results from either the oxidized or reduced AcPAz with AcN2OR. Therefore, results are discussed in terms of one model. In the top 500 complexes

Fig. 2. Cyclic voltammograms (CV) of AcPAz in the presence of MV-activated AcN2OR and as-purified AcN2OR. CVs of AcPAz (100 μM) with MV-activated AcN2OR (1 μM, green dash) and with as-purified AcN2OR (1 μM, red bold) in the presence of saturated N2O at pH 6.0, 25 °C, and in 100 mM phosphate under anoxic conditions. CVs were recorded at a scan rate of 2.0 mV/s. The dotted line (−−−) represents AcPAz with as-purified AcN2OR in the absence of N2O.

K. Fujita et al. / Journal of Inorganic Biochemistry 115 (2012) 163–173

ranked by global score, AcPAz docked with AcN2OR at specific surfaces close to the CuA centers (Fig. S1A). We also confirmed previous results that cytc552 docked close to the CuA centers in MhN2OR (Fig. S1B) [30]. The docking locations of the non-physiological electron donor, BHcytc were scattered on the surface of AcN2OR (Fig. S1C) indicative of less specific interactions between the two proteins. The majority of the lower ranked global score complexes of BHcytc– AcN2OR converged at the groove of N2OR between the Cu centers, consistent with the observation from HHcytc (cytochrome c from horse heart) with MhN2OR [30].

167

The top 20 candidate complexes were generated by selecting from the top 500 globally scored complexes based on the distance of less than 20 Å between CuA center of N2ORs and the redox center of the ET donors, and evaluated using the PROTORP server [43], as summarized in Table 3 for reduced AcPAz–AcN2OR and Supplemental Table 1 for cytc552–MhN2OR. The ratio of nonpolar residues on the interface (50.4 ± 4.4%), reflecting the hydrophobicity of the interface region [53,54], suggests a relatively minor but perhaps significant contribution from hydrophobic interactions to the formation of the protein complex for AcPAz–AcN2OR (see Discussion). Other parameters (Table 3) are broadly consistent with those for cytc552–MhN2OR (Table S1). Prediction of the most favorable ET routes from AcPAz to AcN2OR was accomplished using the PATHWAYS algorithm [46,47]. The top 20 pathways exhibit relatively high electronic coupling constants, HDA (from 199.6 × 10 −6 to 1.2 × 10 −6), compared to the HDA values from cytc552 to MhN2OR (from 7.6 × 10 −6 to 0.1 × 10 −6) in Table 3 and Supplemental Table 1. Moreover, there are some common residues, such as Leu578 and His576 in N2OR in the most favorable pathways for the AcPAz–AcN2OR complex, while none were evident in the case of cytc552–MhN2OR.

3.4. Reduction of AcN2OR by reduced AcPAz Reduction of oxidized AcN2OR with reduced AcPAz was monitored by UV–vis. Spin-quantifications of the samples were also calculated based on EPR measurements in order to confirm whether a semioxidized AcN2OR state ([2Cu(II)·2Cu(I)] in CuZ and [2Cu(I)] in CuA)

Fig. 3. Cyclic voltammograms (CV) of the pH and concentration dependence of as-purified AcN2OR. A, CV plot of the catalytic current as a function of pH. Values were obtained from the CVs of PAz (100 μM) in the presence of a catalytic amount of as-purified AcN2OR (1 μM) and under N2O saturated conditions. B, CVs of PAz (100 μM) in the presence of as-purified AcN2OR as a function of AcN2OR concentration (N2O saturated, 25 °C). CVs were recorded at a scan rate of 2.0 mV/s. The blue solid, green dash, orange dot and red dash–dot lines represent 2.0, 1.0, 0.5 and 0.3 μM N2OR, respectively. C, Catalytic currents plotted as a function of a square-root of the N2OR concentrations. The linear relationship yielded kET =(3.98±0.04)×105 M−1 s−1.

Fig. 4. Cyclic voltammograms (CV) of the inhibition behavior of AcPAz with as-purified AcN2OR in the presence of reduced MV over time. A, CVs of PAz (100 μM) with as-purified N2OR (0.5 μM) and saturated N2O after 1 min (red—dash), 3 min (green— dot), 10 min (blue—dash dot) and 60 min (dark cyan–short dash) incubation with reduced MV (1 μM). The black-bold and gray lines represent the CVs of no MV and PAz only, respectively. B, The catalytic currents (read at 200 mV) plotted as a function of incubation time (k=0.23±0.06 min−1). All CVs were recorded at a scan rate of 2.0 mV/s.

~63 12.9 ± 1.0

It shows the experimental condition and the result with 60-min incubation time after the addition of MV. The experiments were performed after prolonged incubation times (0–60 min) as depicted in Fig. 4.

PAz:N2OR Activity

PAz (μM) N2OR (nM) MV (μM) Incubation time (min) N2OR form Catalysis

a

As-purified 100 500 1 60a 200:1

As-purified 100 500 0 0 200:1 Catalytic current (nA) 194 ± 21 MV-activated 130 4.7 2 0 26,000:1 Specific activity (U/mg) 77.4 ± 5.8

MV-activated 100 500 1 0 200:1

Electrochemistry Solution Scheme

Assay

Table 2 Summary of the experimental parameters for solution and electrochemistry investigations.

~65

K. Fujita et al. / Journal of Inorganic Biochemistry 115 (2012) 163–173 MV-activated 100 1000 ~4 0 100:1

168

could be observed [55]. The addition of reduced AcPAz to oxidized AcN2OR resulted in the observation of the re-oxidation of reduced AcPAz (Fig. 5A). After the addition of reduced AcPAz (20-fold) to oxidized AcN2OR, AcN2OR was separated from an excess amount of AcPAz, followed by a measurement of UV–vis spectrum, Fig. 5B. This spectrum is reminiscent of a mixture of the oxidation states from oxidized AcN2OR ([2Cu(I)·2Cu(II)] in CuZ and [Cu(I)·Cu(II)] in CuA) and as-purified AcN2OR (predominantly [3Cu(I)·Cu(II)] in CuZ and [2Cu(I)] in CuA). The difference spectrum of oxidized AcN2OR with AcPAz-reduced AcN2OR resembles the spectrum of CuA domain of cytochrome c oxidase [56] or CuA-reconstituted AcN2OR in Fig. S3. The EPR spectrum of AcPAz-reduced AcN2OR shows g// = 2.194 and g⊥ = 2.049 with 38G of A// at 10K in Fig. 5C is similar to oxidized AcN2OR. Spin quantification supports that AcPAz-reduced AcN2OR has 1.14 spins per subunit, consistent with a mixture of oxidation stated for AcN2OR [55]. These results further demonstrate that reduced AcPAz is capable of reducing oxidized AcN2OR. 3.5. Physiological concentrations of AcN2OR and AcPAz We estimated the physiological concentrations of AcPAz and AcN2OR in both Ac/pMLnos and wild-type Ac using densitometry analysis from Western blots. In Ac/pMLnos, the amount of AcN2OR expressed was significantly higher than that of AcPAz, as expected. The molar ratio of [AcPAz]/[AcN2OR] was quantified at 0.36 ±0.1 (Table 4 and Fig. 6), and consistent with the overexpression of AcN2OR in Ac/pMLnos. However, in wild type Ac, the ratio of [AcPAz] to [AcN2OR] was significantly higher (40.00 ±2.74, Table 4 and Fig. 6). We acknowledge that the in vitro conditions for the kinetics experiments ([AcPAz]/[AcN2OR] 26,000:1, Table 2) and electrochemistry experiments ([AcPAz]/ [AcN2OR] 200:1 Table 2) were performed at non-physiological relevant ratios; importantly, AcN2OR turnover is still supported. 4. Discussion Spectroscopic, kinetics, and electrochemical data presented within support our hypothesis that PAz is the physiological electron donor to N2OR in Ac. We outline the evidence in this section under the following subheadings: electrochemistry and kinetics; electron transfer outcomes; and mechanistic considerations of AcN2OR and AcPAz. We conclude the discussion with a section on the physiological relevance of our results and related findings from other groups. 4.1. Electrochemistry and kinetics of AcN2OR and AcPAz The spectroscopic, kinetics and electrochemical experiments strongly support that AcPAz is a physiological electron donor for AcN2OR. Interestingly, substantially diminished catalytic activity was observed with PAz and MV-activated AcN2OR (Fig. 2). This result differs from the enhanced catalytic response from the as-purified AcN2OR (Figs. 2 and 3). In addition, it is clear that MV inhibits the solution reaction between AcN2OR and AcPAz at lower concentrations of AcPAz (Table 2). We believe these results reveal an inhibitory effect of reduced MV in the reaction of activated AcN2OR with AcPAz. Notably, previous reports established that MV did not inhibit electron transfer between N2OR and BV (up to 1 mM [19]). These results collectively suggest that MV influences the interaction between AcPAz and AcN2OR. One possibility is that a relatively tight association of MV with AcN2OR could facilitate electron donation by MV in the activation reaction and interfere in the intermolecular ET between AcPAz and AcN2OR during turnover. It should be noted that a loss of the ET ability of AcPAz by MV can be excluded because no change was observed in electrochemical measurements of PAz in the presence of MV. These results may be related to previously documented turnover dependent inactivation of N2OR owing to either MV or BV [57–59]. The observations that catalytic turnover is supported in

K. Fujita et al. / Journal of Inorganic Biochemistry 115 (2012) 163–173

experiments when AcPAz is present in a large excess (Table 2) suggest that MV inhibition may be reversible. 4.2. Electron transfer outcomes The overall docking outcome for the AcPAz–AcN2OR complex is in good agreement with that for cytc552–MhN2OR (Figs. S1A and S1B), suggesting that AcPAz and cytc552 interact with their respective N2ORs in a closely similar manner, and consistent with the role of CuA as the electron entry point to the enzymes. These findings broadly agree with the recent results which were performed using the same algorithm generated independently by Dell'Acqua et al. [60]. On the other hand, BHcytc–AcN2OR has a significantly lower average global score (2.2± 0.2) than the others indicative of non-physiological interactions (Table S2). This observation is entirely consistent with the kinetics and electrochemistry data. Based on interface analyses and the docking parameters, both the AcPAz–AcN2OR and cytc552–MhN2OR complexes may be characterized as transient complexes [53,61,62], consistent with expectations for facile electron transfer. The nonpolar ratio (~ 50%) we calculated for the AcPAz–AcN2OR complex is fairly similar to that calculated herein for cytc552– MhN2OR. Ratios of this magnitude do not permit an unambiguous determination of whether hydrophobic interactions control the protein–protein interaction, as discussed by Dell'Acqua et al. [30]. Sorting the best structures for cytc552–MhN2OR from our calculations by the method described [30] increased the ratio to 57.8–71.4% (65.5 ± 4.8%), in good agreement with their result. Interestingly, when this analysis was applied to AcPAz–AcN2OR, the ratio was unchanged 52.3–57.3% (54.0 ± 1.7%), perhaps implying that hydrophobic interactions are relatively less important between AcPAz and AcN2OR than between cytc552 and MhN2OR. It is also useful to compare the predicted intermolecular ET pathways (based on values of HDA) for AcPAz–AcN2OR and cytc552–MhN2OR. Most of the routes in AcPAz–AcN2OR span only 3 to 4 residues, whereas those in

169

cytc552–MhN2OR utilize 4–6 residues. The top 7 complexes of AcPAz– AcN2OR exhibit HDA values of ≈1.2×10−5 to 2.0×10−4 whereas the values for cytc552–MhN2OR are generally lower (Tables 3 and S1). Interestingly, these predictions mirror the kcat differences; AcPAz–AcN2OR (89.3 s−1) versus cytc552–MhN2OR (3.8 s−1) [30]. Fig. 7 illustrates the representative ET pathways in the top 3 complexes based on HDA. As seen in Table 3, the PATHWAYS analysis suggests a primary electron accepting residue in AcN2OR along the ET path is either His576 or Leu578 and both of the residues are well conserved among N2ORs [10,25]. Mattila et al. proposed that His635 (corresponding to His576 in AcN2OR) is involved in the ET pathway based on their docking simulations between P. pantotrophus PAz (PpPAz) and PdN2OR which is phylogenetically related to AcN2OR [2,25]. In addition, Dell'Acqua et al. suggested that the ET pathway involving His566 or Leu568 in MhN2OR (corresponding to His576 or Leu578 in AcN2OR, respectively) is the most plausible ET route [60] which is consistent with our predictions. It should be noted that Asp588 (533 in AcN2OR), proposed as a key interfacial residue in the docking of PdN2OR with PpPAz [25], was not assigned as a part of ET routes using the PATHWAYS algorithm with our protocol. Pomowski et al. reported that His583 in P. stutzeri N2OR (PsN2OR), corresponding to His536 in AcN2OR, was tilted by ca. 130° and not coordinated to one of Cu atom in CuA in the anoxic isolated form I [16]. However, once N2O was bound near CuZ, His583 became coordinated to CuA. Interestingly, the analogous histidine (His536 in AcN2OR) bound to the same Cu atom in CuA as His579 in AcN2OR is proposed to be involved in the ET pathways (Fig. 7). Collectively, the structural data coupled with our calculations suggest to us to propose that the motion of His583 (His536 in AcN2OR) may regulate the redox potential of CuA to facilitate the multi-electron reduction of CuZ. 4.3. Mechanistic considerations It is interesting that both very strong reductants (the viologens) and mild reductants (the physiological partners) comparably support turnover of AcN2OR. The KM (N2O) values for AcN2OR are essentially

Table 3 Interface analyses of the candidate complexes of PAz and AcN2OR. Global scorea

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 Ave sd

2.5 2.7 5.4 2.5 2.7 2.7 2.7 2.8 2.5 2.5 3.9 2.5 2.4 3.3 2.5 4.8 3.1 3.4 2.7 4.3 3.1 0.9

Hydrophobic Interface area, scorea ΔASA (Å2)

−4.5 −3.7 −5.0 −4.2 −4.4 −3.5 −5.1 −5.1 −5.3 −3.5 −3.7 −4.5 −3.6 −4.4 −4.2 −4.3 −4.3 −4.5 −5.4 −5.4 −4.4 0.6

908 938 880 973 887 962 945 889 954 975 950 1008 816 834 910 1011 915 961 963 929 930 52

b

Gap vol index, b (Å)

3.0 3.3 3.3 2.5 3.2 3.0 3.5 2.8 3.0 3.1 2.4 3.1 3.3 3.2 3.4 2.9 3.4 3.4 3.3 3.5 3.1 0.3

Nonpolar ratio, b (%)

55.2 51.2 57.3 48.3 52.1 52.3 39.6 52.6 48.4 48.8 52.3 52.6 43.0 50.4 54.6 53.7 53.8 44.0 50.2 47.8 50.4 4.4

Planarity, CuA– Cu(PAz) (Å) (Å)

Electron transfer pathway

b

2.2 1.8 2.2 2.1 2.1 2.1 1.7 2.0 1.8 1.8 2.0 1.9 1.7 1.8 1.9 2.0 1.9 1.8 1.9 1.8 1.9 0.1

13.8 15.5 15.0 18.5 14.9 16.6 17.7 17.8 18.4 18.2 18.1 18.3 16.2 17.9 17.8 17.9 17.6 15.8 18.5 19.1 17.2 1.5

Coupling constant, HDA, c by 10−6

Number of residues

Throughspace jump Interfacial path (PAz-N2OR)

Interfacial distance, d (Å)

199.6 172.6 65.8 35.7 25.7 13.5 11.5 9.7 9.4 8.7 7.3 5.5 5.4 4.8 4.0 3.8 2.7 2.4 2.3 1.2 –

3 3 3 4 4 4 3 4 4 4 4 5 4 4 4 4 4 4 4 5 3.9 0.6

His81-His576 His81-His576 His81-Leu578 Pro80-Leu578 Pro80-His576 Thr79-His576 His81-Leu578 Met84-Leu578 Pro80-Leu578 Thr79-His576 Pro80-Leu578 Gly83-Leu578 Pro80-Leu578 Met84-Leu578 Met84-His576 Tyr82-His576 Tyr82-His576 Pro80-His576 Met84-Leu578 Pro80-Ala577 –

2.5 2.3 3.2 2.0 2.5 2.6 3.9 2.0 2.8 2.8 2.9 2.2 3.1 1.8 1.5 2.4 3.2 3.3 2.0 4.0 2.7 0.7

Multiple throughspace paths are involved in the total ET route. a Generated by the BiGGER algorithm in Ref. [41]. b Estimated by the protein–protein interface analysis server, PROTORP in Ref. [43] and ΔASA, gap volume index, nonpolar ratio and planarity defined in Refs. [44,45]. c Calculated using the PATHWAYS algorithm in Refs. [46,47], implemented in molecular modeling package, HARLEM in Ref. [48]. d The interfacial distance was defined as the atomic distance between the atom on the donor molecular surface and the other on the acceptor molecular surface.

170

K. Fujita et al. / Journal of Inorganic Biochemistry 115 (2012) 163–173

Fig. 5. Reduction of oxidized AcN2OR by reduced AcPAz. A, Titration of oxidized AcN2OR with ~10 equivalents of reduced AcPAz monitored by UV–vis spectra. B, Spectrum of AcN2OR reduced by reduced AcPAz (black—solid line) after separation of excess AcPAz in comparison with spectra of oxidized AcN2OR (magenta—dash line) and as-purified AcN2OR (blue—dot line). UV–vis spectra were measured at 25 °C in 50 mM phosphate, pH 7.1 under anoxic conditions. C, EPR spectra of AcN2OR reduced by reduced AcPAz (bold line) and oxidized AcN2OR (red—dash line) as isolated in 50 mM phosphate, pH 7.1. Spectrometer configurations: microwave power, 1.6 mW; frequency, 9.34 GHz; temperature, 10 K; modulation amplitude, 0.5 mT; modulation frequency, 100 kHz; sweep rate, 0.2 mT s−1.

identical for turnover with AcPAz and BV (19.1 μM for AcPAz vs 24.9 μM for BV) [17]. kcat is more sensitive to the identity of the electron donor: kcat = 162.9 s −1 with BV but kcat = 89.3 s −1 with AcPAz [17]. This modest effect may be attributable to differences in active-site accessibility. AcPAz likely accesses a limited surface of AcN2OR to transfer electrons to CuZ via CuA, whereas a small molecule such as BV may react with both CuA and directly with CuZ in AcN2OR. This interpretation is also supported by the differences observed in the KM values for AcPAz (28.8 μM) and BV (13.8 μM, see Fig. S2). In contrast, turnover by MhN2OR appears to be more sensitive to reductant: a specific activity of 157 U/mg was observed with MV versus 1.8 U/mg with cytc552 (Table 1) [30].

Other interesting distinctions in the behavior between MhN2OR and AcN2OR exist. In contrast to our results, KM is larger for the protein donor to MhN2OR; 50.2 μM with respect to cytc552, compared to 11.5 μM with respect to MV ([N2O] = 1.25 mM) (see Figs. 1C, S2, and Table 1) [30]. AcPAz-supported catalysis by AcN2OR is dependent upon the concentration of N2O. In contrast, the catalytic turnover of MhN2OR mediated by cytc552 is independent of N2O concentration [30]. Further, the electrochemical behavior of MhN2OR with cytc552 is also different from that of AcN2OR with AcPAz [52]. These findings suggest that turnover mediated by the physiological electron donors may differ between AcN2OR and MhN2OR despite their close structural similarity. In contrast, the viologen-mediated turnover by both N2ORs displays a similar dependence on the concentration of N2O and comparable KM values (Table 1). Finally, it should be noted that the kcat for air-purified MV-activated MhN2OR (321 s −1) is approximately twice that of the anoxic purified MV-activated AcN2OR (163 s −1) [17,30]. Our results, together with data from other systems [52,63] indicate that reductants are likely to have two distinct roles in N2OR catalysis (Scheme 1). It is clear that a low potential reductant is required to prepare the activated state, CuZ = [4Cu(I)]. Reaction with either reduced MV or BV (− 441 and − 360 mV, vs. NHE respectively) [64] can produce fully-reduced CuZ when added to the as-purified enzyme. The CuZ = [4Cu(I)] state has been suggested to be the state that reacts with N2O to reduce it to N2. In light of our present data, and numerous other considerations [17–19,21,30,52], if fully-reduced CuZ is a catalytic intermediate, turnover would have to be supported by reductants with substantially higher redox potentials, e.g. the physiological partners identified here and elsewhere. As illustrated in Scheme 1, one possibility is that the redox states of CuZ in the key catalytic intermediates are [2Cu(II)·2Cu(I)] and [4Cu(I)]. Mild, physiological reductants such as PAz and cytc552 are capable of reducing the [2Cu(II)·2Cu(I)] state present during catalysis to the fully-reduced state of CuZ, which then reduces N2O, because the ET event between CuA and CuZ is driven by the reduction of N2OR by reduced PAz (see Fig. 5). If this is the case, the data would further indicate that the catalytic [2Cu(II)·2Cu(I)] state of CuZ must differ from the CuZ states present in the “as-isolated” form, since the latter require much more powerful reductants to generate the [4Cu(I)] state. In MhN2OR, Dell'Acqua et al. [52] identified a new intermediate (CuZ°) as the key reactive species in catalysis. CuZ° corresponds to species B ([3Cu(I)·Cu(II)]) illustrated in our scheme. Since PAz is a one-electron reductant, both A and B (Scheme 1) are plausible catalytic intermediates. The rates of formation of A, and its reduction to B, and the subsequent reduction of B, may vary among N2OR enzymes. Thus, the relative concentrations of A and B (CuZ°) could also depend upon the identity of the specific N2OR examined. The time dependent inhibition of electron transfer by MV between AcPAz and AcN2OR (k = 0.23 min −1, Fig. 4B) is in excellent agreement with that of the inactivation process of cytc552–MhN2OR (k = 0.30 min −1) in the absence of reductants. Dell'Acqua et al. reported separating MV from N2OR using gel filtration. However, it is still possible that low levels of residual MV may be present in their experiments. Table 4 Estimated physiological concentrations of PAz and N2OR in Ac/pMLnos and in wild-type Ac. Strain pMLnos

Wild-type

[PAz] (μM)

[N2OR] (μM)

[PAz]/ [N2OR]

[PAz] (μM)

[N2OR] (μM)

[PAz]/ [N2OR]

9.10 11.19 11.65

28.01 23.63 41.98

0.32 0.47 0.28 0.36 ± 0.10

28.74 15.71 21.98

0.68 0.38 0.60

42.15 40.89 36.90 40.00 ± 2.74

K. Fujita et al. / Journal of Inorganic Biochemistry 115 (2012) 163–173

171

Fig. 6. Western blots of N2OR and PAz expression in wild-type Ac and Ac/pMLnos. A, AcN2OR: lane 1 mw-marker, lanes 2–4 Ac/pMLnos, lanes 5–7 wild-type Ac, lanes 8–12 represent 4, 7, 14, 28, and 57 ng respectively of AcN2OR. B, PAz; lane 1 mw-marker; lanes 2–7 represent 356, 178, 89, 45, 22, and 11 ng of AcPAz respectively, lanes 8–10 and 11–13 represent 8× dilution of wt and pMLnos, supernatant.

4.4. Physiological relevance in denitrification The kinetics and electrochemical data establish that AcPAz supports catalytic N2O reduction in vitro. These observations are strongly supported by the absence of spectral and electrochemical changes with Accytc555, a potential alternative electron donor, and BHcytc, a non-physiological electron donor. Further, the kinetics results and the weak binding between AcPAz and AcN2OR imply that an AcPAz– AcN2OR complex is structured to facilitate intermolecular electron transfer from AcPAz to AcN2OR. Quantitative immunoblots of wildtype Ac clearly support that AcPAz is expressed at a relatively higher level than that of AcN2OR. AcPAz has been identified as an electron donor of NiR, which is also involved in the denitrification pathway [28,29]. On the assumption that PAz functions as an in vivo electron donor towards both N2OR and NiR, the different pH dependences of the reactions of PAz with the enzymes may have regulatory implications in a pathway coupled to H + translocation. Given the results reported herein, and those reported previously [30,52], either a blue copper protein or a cytochrome may function as the electron donor to N2OR depending on the organism. In Pd,

both cytc550 and a blue copper protein are involved as electrontransfer proteins in the denitrification pathway [22–24]. In Mh, cytc552 was identified in donating electrons to not only cytochrome cd1 [31] and nitric oxide reductase [65], but also to N2OR [30]. Consequently, the question arises as whether Ac also has a c-type cytochrome that may serve as an alternative electron donor to N2OR. We have observed multiple c-type cytochromes in Ac, and isolated Accytc555. Accytc555 is dominantly expressed amongst observed c-type cytochromes at least under the growth conditions in our investigation (see Supplemental information), No support of AcN2ORcatalysis was exhibited by Accytc555. This result contrasts with both cytc550 and PAz involved in denitrifying reactions with Pd under in vivo conditions [22]. However, the possibility of other cytochromes in Ac donating electrons to N2OR cannot be ruled out. It is worth noting that in contrast to what we observe with BHcytc and AcN2OR, Rasmussen et al. reported rapid electron transfer between HHcytc and N2OR from P. pantotrophus (PpN2OR) and observed a single turnover by PpN2OR in the presence of N2O and reduced HHcytc [63]. Apparently, N2ORs from different organisms may differ with regard to their specificity and selectivity for electron transfer.

Fig. 7. Representative ET pathways from the Type 1 Cu of AcPAz to the CuA center of AcN2OR. The top three pathways are shown in different colors: model 1 (orange), model 2 (green) and model 3 (blue). Key features of the complex include the CuA center (yellow spheres), and residues His576, Cys575 and Leu578 and His579. Type 1 Cu and the corresponding His81 ligand are identified by the respective model colors. Dashed lines indicate the through space jump. The inset shows the superposition of the globular structures of three PAz–AcN2OR complexes.

172

K. Fujita et al. / Journal of Inorganic Biochemistry 115 (2012) 163–173

Ps 4-pyds TAPS

Pseudomonas stutzeri 4,4-dithiodipyridine N-tris(hydroxymethyl)methyl-3-aminopropanesulfonic acid

Acknowledgment We thank Professor R. Szilagyi for the valuable computational discussions. We acknowledge Professor J. Ponder and his colleagues for creating the Tinker 4.2 software. This work was supported by the National Science Foundation, MCB-0744289 to D.M.D., a Grant-in-Aid for Scientific Research from JSPS, No. 18550147, Japan to T.K. and the Project of Development of Basic Technologies for Advanced Production Methods Using Microorganism Functions by NEDO to T.K. Scheme 1. Proposed catalytic mechanism of electron transfer from PAz to AcN2OR.

5. Conclusions AcPAz is clearly capable of supporting the catalytic reduction of N2O as an electron donor in vitro; in addition it binds weakly to AcN2OR. It is therefore probable that PAz is the physiological electron donor for N2OR in Ac. There may be two distinct roles for reductants in N2OR catalysis in vivo, as established for in vitro conditions: (i) to generate the catalytically-active CuZ = [4Cu(I)] state, and (ii) to reduce the CuZ = [2Cu(II)·2Cu(I)] produced by the reduction of N2O in the catalytic cycle (Scheme 1). Moreover, pH dependence of the electrochemical reaction exhibits an optimal condition (pH ~ 6) but is a bit more acidic than that of the ET reaction between AcPAz and AcNiR (pH > 7) [29]. Docking studies indicate that PAz interacts with the N2OR surface near the CuA center and multiple, favorable ET pathways between PAz and AcN2OR are available. 6. Abbreviations

Ac AcN2OR Accytc555 AcPAz BCA BCIP BHcytc BSA BV CHES cytc550 cytc552 DT ET Hd HHcytc KM Mh MES MOPS MV NBT N2OR NiR Pd Pp PAz

Achromobacter cycloclastes Achromobacter cycloclastes nitrous oxide Reductase Achromobacter cycloclastes cytochrome c555 Achromobacter cycloclastes pseudoazurin bicinchoninic acid 5-bromo-4-chloro-3-indolyl phosphate bovine heart cytochrome c bovine serum albumin benzyl viologen (4,4′-dibenzylpyridinium dichloride) 2-(cyclohexylamino)ethanesulfonic acid cytochrome c550 cytochrome c552 sodium dithionite electron transfer Hyphomicrobium denitrificans horse heart cytochrome c Michaelis constant Marinobacter hydrocarbonclasticus 2-(N-morpholino)ethanesulfonic acid 3-(N-morpholino)propanesulfonic acid methyl viologen (4,4′-dimethylpyridinium dichloride) nitroblue tetrazolium nitrous oxide reductase nitrite reductase Paracoccus denitrificans Paracoccus pantotrophus pseudoazurin

Appendix A. Supplementary data Supplementary data to this article can be found online at http:// dx.doi.org/10.1016/j.jinorgbio.2012.07.013. References [1] W.G. Zumft, P.M.H. Kroneck, Adv. Microb. Physiol. 52 (2007) 109–227. [2] W.G. Zumft, Microbiol. Mol. Biol. Rev. 61 (1997) 533–616. [3] P. Crutzen, in: C. Delwiche (Ed.), Denitrification, Nitrification and Atmospheric Nitrous Oxide, John Wiley, New York, 1981, pp. 17–44. [4] R.E. Dickinson, Nature 319 (1986) 109–115. [5] W.L. Jolly, The Inorganic Chemistry of Nitrogen, W. A. Benjamin, New York, 1964. [6] M.H. Thiemens, W.C. Trogler, Science 251 (1991) 932–934. [7] A.R. Ravishankara, J.S. Daniel, R.W. Portmann, Science 326 (2009) 123–125. [8] K. Brown, M. Tegoni, M. Prudencio, A.S. Pereira, S. Besson, J.J. Moura, I. Moura, C. Cambillau, Nat. Struct. Biol. 7 (2000) 191–195. [9] K. Brown, K. Djinovic-Carugo, T. Haltia, I. Cabrito, M. Saraste, J.J.G. Moura, I. Moura, M. Tegoni, C. Cambillau, J. Biol. Chem. 275 (2000) 41133–41136. [10] T. Haltia, K. Brown, M. Tegoni, C. Cambillau, M. Saraste, K. Mattila, K. Djinovic-Carugo, Biochem. J. 369 (2003) 77–88. [11] K. Paraskevopoulos, S.V. Antonyuk, R.G. Sawers, R.R. Eady, S.S. Hasnain, J. Mol. Biol. 362 (2006) 55–65. [12] P.M.H. Kroneck, W.A. Antholine, J. Riester, W.G. Zumft, FEBS Lett. 248 (1989) 212–213. [13] R.A. Scott, W.G. Zumft, C.L. Coyle, D.M. Dooley, Proc. Natl. Acad. Sci. U. S. A. 86 (1989) 4082–4086. [14] T. Rasmussen, B.C. Berks, J. Sanders-Loehr, D.M. Dooley, W.G. Zumft, A.J. Thomson, Biochemistry 39 (2000) 12753–12756. [15] M.L. Alvarez, J. Ai, W. Zumft, J. Sanders-Loehr, D.M. Dooley, J. Am. Chem. Soc. 123 (2001) 576–587. [16] A. Pomowski, W.G. Zumft, P.M.H. Kroneck, O. Einsle, Nature 477 (2011) 234–237. [17] K. Fujita, J.M. Chan, J.A. Bollinger, M.L. Alvarez, D.M. Dooley, J. Inorg. Biochem. 101 (2007) 1836–1844. [18] S. Ghosh, S.I. Gorelsky, P. Chen, I. Cabrito, J.J.G. Moura, I. Moura, E.I. Solomon, J. Am. Chem. Soc. 125 (2003) 15708–15709. [19] J.M. Chan, J.A. Bollinger, C.L. Grewell, D.M. Dooley, J. Am. Chem. Soc. 126 (2004) 3030–3031. [20] K. Fujita, D.M. Dooley, Inorg. Chem. 46 (2007) 613–615. [21] S. Dell'Acqua, S.R. Pauleta, I. Moura, J.J.G. Moura, J. Biol. Inorg. Chem. 16 (2011) 183–194. [22] M. Koutny, I. Kučera, R. Tesařík, J. Turánek, R.J.M. Van Spanning, FEBS Lett. 448 (1999) 157–159. [23] J.W.B. Moir, S.J. Ferguson, Microbiol. 140 (1994) 389–397. [24] I.V. Pearson, M.D. Page, R.J.M. Van Spanning, S.J. Ferguson, J. Bacteriol. 185 (2003) 6308–6315. [25] K. Mattila, T. Haltia, Proteins 59 (2005) 708–722. [26] S.R. Pauleta, F. Guerlesquin, C.F. Goodhew, B. Devreese, J. Van Beeumen, A.S. Pereira, I. Moura, G.W. Pettigrew, Biochemistry 43 (2004) 11214–11225. [27] B.C. Berk, D. Baratta, D.J. Richardson, S.J. Ferguson, Eur. J. Biochem. 212 (1993) 467–476. [28] M.A. Kashem, H.B. Dunford, M.Y. Liu, W.J. Payne, J. LeGall 145 (1987) 563–568. [29] T. Kohzuma, S. Takase, S. Shidara, S. Suzuki, Chem. Lett. (1993) 149–152. [30] S. Dell'Acqua, S.R. Pauleta, E. Monzani, A.S. Pereira, L. Casella, J.J.G. Moura, I. Moura, Biochemistry 47 (2008) 10852–10862. [31] H. Lopes, S. Besson, I. Moura, J.J.G. Moura, J. Biol. Inorg. Chem. 6 (2001) 55–62. [32] L.M. Saraiva, S. Besson, I. Moura, G. Fauque, Biochem. Biophys. Res. Commun. 212 (1995) 1088–1097. [33] G.M.J. Fauque, S. Besson, L. Saraiva, I. Moura, Oceanis 18 (1992) 211–216. [34] D. Hira, M. Nojiri, K. Yamaguchi, S. Suzuki, J. Biochem. 142 (2007) 335–341. [35] Deligeer, R. Fukunaga, K. Kataoka, K. Yamaguchi, K. Kobayashi, S. Tagawa, S. Suzuki, J. Inorg. Biochem. 91 (2002) 132–138.

K. Fujita et al. / Journal of Inorganic Biochemistry 115 (2012) 163–173 [36] K. Yamaguchi, A. Kawamura, H. Ogawa, S. Suzuki, J. Biochem. 134 (2003) 853–858. [37] R.F. Abdelhamid, Y. Obara, Y. Uchida, T. Kohzuma, D.M. Dooley, D.E. Brown, H. Hori, J. Biol. Inorg. Chem. 12 (2007) 165–173. [38] P.K. Smith, R.I. Krohn, G.T. Hermanson, A.K. Mallia, F.H. Gartner, M.D. Provenzano, E.K. Fujimoto, et al., Anal. Biochem. 150 (1985) 76–85. [39] T. Kohzuma, C. Dennison, W. McFarlane, S. Nakashima, T. Kitagawa, T. Inoue, Y. Kai, N. Nishio, S. Shidara, S. Suzuki, A.G. Sykes, J. Biol. Chem. 270 (1995) 25733–25738. [40] L.A. Coury Jr., B.N. Oliver, J.O. Egekeze, C.S. Sosnoff, J.C. Brumfield, R.P. Buck, R.W. Murray, Anal. Chem. 62 (1990) 452–458. [41] P.N. Palma, L. Krippahl, J.E. Wampler, J.J.G. Moura, Proteins 39 (2000) 372–384. [42] P. Ren, J.W. Ponder, J. Comput. Chem. 23 (2002) 1497–1506. [43] C. Reynolds, D. Damerell, S. Jones, Bioinformatics 25 (2009) 413–414. [44] L.A. Laskowski, J. Mol. Graph. 13 (1995) 323–330. [45] B. Lee, F.M. Richards, J. Mol. Biol. 55 (1971) 379–400. [46] J.N. Betts, D.N. Beratan, J.N. Onuchic, J. Am. Chem. Soc. 114 (1992) 4043–4046. [47] J.J. Regan, S.M. Risser, D.N. Baratan, J.N. Onuchic, J. Phys. Chem. 97 (1993) 13083–13088. [48] I.V. Kurnikov, M.G. Kurnikova, W. Wenzel, HARLEM — Biomolecular Modeling Package, http://www.kurnikov.org/harlem_main.html. [49] T. Ferreira, W. Rasband, The ImageJ User Guide— Version 1.43, http://rsbweb.nih. gov/ij/docs/user-guide.pdf. [50] L.M. Saraiva, A.J. Thomson, N.E. Le Brun, M.-Y. Liu, W.J. Payne, J. LeGall, I. Moura, Biochem. Biophys. Res. Commun. 204 (1994) 120–128.

173

[51] A.J. Bard, L.R. Faulkner, Electrochemical Methods: Fundamentals and Applications, Wiley, New York, 1980. [52] S. Dell'Acqua, S.R. Pauleta, P.M.P. de Sousa, E. Monzani, L. Casella, J.J.G. Moura, I. Moura, J. Biol. Inorg. Chem. 15 (2010) 967–976. [53] S. Jones, J.M. Thornton, Proc. Natl. Acad. Sci. U. S. A. 93 (1996) 13–20. [54] L.L. Lo Conte, C. Chothia, J. Janin, J. Mol. Biol. 285 (1999) 2177–2198. [55] J.A. Farrer, A.J. Thomson, M.R. Cheesman, D.M. Dooley, W.G. Zumft, FEBS Lett. 294 (1991) 11–15. [56] C. von Wachenfeldt, S. de Vries, J. van der Oost, FEBS Lett. 340 (1994) 109–113. [57] C.L. Coyle, W.G. Zumft, P.M.H. Kroneck, H. Körner, W. Jakob, Eur. J. Biochem. 153 (1985) 459–467. [58] S.W. Snyder, T.C. Hollocher, J. Biol. Chem. 262 (1987) 6515–6525. [59] C.K. SooHoo, T.C. Hollocher, J. Biol. Chem. 266 (1991) 2203–2209. [60] S. Dell'Acqua, I. Moura, J.J.G. Moura, S.R. Pauleta, J. Biol. Inorg. Chem. 16 (2011) 1241–1254. [61] I.M.A. Nooren, J.M. Thornton, J. Mol. Biol. 325 (2003) 991–1018. [62] I.M.A. Nooren, J.M. Thornton, EMBO J. 22 (2003) 3486–3492. [63] T. Rasmussen, T. Brittain, B.C. Berks, N.J. Watmough, A.J. Thomson, Dalton Trans (2005) 3501–3506. [64] G.D. Watt, A. Burns, Biochem. J. 152 (1975) 33–37. [65] C.E. Martins, A.S. Pereira, P. Tavares, C.M. Cordas, F. Folgosa, C.G. Timoteo, S. Naik, B.H. Huynh, J.J.G. Moura, I. Moura, J. Biol. Inorg. Chem. 12 (2007) S53–S98.