Published on Web 03/10/2006
NO Reductase Activity of the Tetraheme Cytochrome c554 of Nitrosomonas europaea Anup K. Upadhyay,† Alan B. Hooper,‡ and Michael P. Hendrich*,† Contribution from the Department of Chemistry, Carnegie Mellon UniVersity, Pittsburgh, PennsylVania 15213, and Department of Biochemistry, UniVersity of Minnesota, St. Paul, Minnesota 55108 Received August 16, 2005; E-mail:
[email protected] Abstract: The tetraheme cytochrome c554 (cyt c554) from Nitrosomonas europaea is believed to function as an electron-transfer protein from hydroxylamine oxidoreductase (HAO). We show here that cyt c554 also has significant NO reductase activity. The protein contains one high-spin and three low-spin c-type hemes. HAO catalyzed reduction of the cyt c554, ligand binding, intermolecular electron transfer, and kinetics of NO reduction by cyt c554 have been investigated. We detect the formation of a NO-bound ferrous heme species in cyt c554 by EPR and Mo¨ssbauer spectroscopies during the HAO catalyzed oxidation of hydroxylamine, indicating that N-oxide intermediates produced from HAO readily bind to cyt c554. In the half-reduced state of cyt c554, we detect a spin interaction between the [FeNO]7 state of heme 2 and the low-spin ferric state of heme 4. We find that ferrous cyt c554 will reduce NO at a rate greater than 16 s-1, which is comparable to rates of other known NO reductases. Carbon monoxide or nitrite are shown not to bind to the reduced protein, and previous results indicate the reactions with O2 are slow and that a variety of ligands will not bind in the oxidized state. Thus, the enzymatic site is highly selective for NO. The NO reductase activity of cyt c554 may be important during ammonia oxidation in N. europaea at low oxygen concentrations to detoxify NO produced by reduction of nitrite or incomplete oxidation of hydroxylamine.
Introduction
The chemoautotrophic soil bacterium, Nitrosomonas europaea, derives energy for growth from the oxidation of ammonia to nitrite. NO is produced by Nitrosomonas by a periplasmic copper-containing nitrite reductase, using nitrite as a terminal electron acceptor under limiting oxygen conditions.1,2 However, the production of NO (and then N2O) during oxidation of ammonia to nitrite under low [O2] in N. europaea has been found to occur in the absence of a functional NirK gene,3 indicating that enzyme(s) other than nitrite reductase also produce NO. The presence of excess NO induces denitrification in nitrifying bacteria.4-8 Recent studies have shown that free NO acts as a signaling molecule in regulating the growth pattern and metabolic activity of N. europaea.9 The presence of excess nitrite10 and NO11 has been found to be toxic to Nitrosomonas. † ‡
Carnegie Mellon University. University of Minnesota.
(1) Abeliovich, A.; Vonshak, A. Arch. Microbiol. 1992, 158, 267-270. (2) Schmidt, I.; Bock, E. Arch. Microbiol. 1997, 167, 106-111. (3) Beaumont, H. J. E.; Hommes, N. G.; Sayavedra-Soto, L. A.; Arp, D. J.; Arciero, D. M.; Hooper, A. B.; Westerhoff, H. V.; Van Spanning, R. J. M. J. Bacteriol. 2002, 184, 2557-2560. (4) Schmidt, I.; Bock, E. Antonie Van Leeuwenhoek 1998, 73, 271-278. (5) Schmidt, I.; Bock, E.; Jetten, M. S. M. Microbiology 2001, 147, 22472253. (6) Schmidt, I.; Hermelink, C.; Van de Pas-Schoonen, K.; Strous, M.; op den Camp, H. J.; Kuenen, J. G.; Jetten, M. S. M. Appl. EnViron. Microbiol. 2002, 68, 5351-5357. (7) Schmidt, I.; Zart, D.; Bock, E. Antonie Van Leeuwenhoek 2001, 79, 3947. (8) Schmidt, I.; Zart, D.; Bock, E. Antonie Van Leeuwenhoek 2001, 79, 311318. (9) Schmidt, I.; Steenbakkers, P. J. M.; op den Camp, H. J. M.; Schmidt, K.; Jetten, M. S. M. J. Bacteriol. 2004, 186, 2781-2788. 4330
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A gene, NorB coding for a membrane cytochrome cbb3 NOreductase is found in the genome of Nitrosomonas.12 Genetic removal of the NorB gene has been shown to strongly diminish the conversion of nitrite to NO and then N2O in cells of Nitrosomonas indicating an important role for the membrane cytochrome cbb3 NO-reductase.13 There are, however, indications that some other catalyst for reduction of NO to N2O might exist; N2O production and resistance to NO toxicity continues in the NorB mutant.13,14 The NO reductase activity of cytochrome c554, shown here, may be a candidate for providing at least a low flux of NO reduction to counter NO toxicity. Cyt c554 is a monomeric protein (26 kDa) found in the periplasm with an R-helical structure containing four c-type hemes covalently bound via two cysteine thioether linkages in the -C-X-Y-C-H- sequence.15 The crystal structure of cyt c554 shows that hemes 1, 3, and 4 have bis-His axial coordination. Heme 2 is five coordinate with one axial His ligand.16,17 The four hemes in cyt c554 are present in two diheme pairs (10) Stein, L. Y.; Arp, D. J. Appl. EnViron. Microbiol. 1998, 64, 4098-4102. (11) Liu, K. E.; Valentine, A. M.; Wang, D.; Huynh, B. H.; Edmondson, D. E.; Salifoglou, A.; Lippard, S. J. NATO ASI Series, Series 2: EnVironment 1997, 26, 273-286. (12) Chain, P.; Lamerdin, J.; Larimer, F.; Regala, W.; Lao, V.; Land, M.; Hauser, L.; Hooper, A.; Klotz, M.; Norton, J.; Sayavedra-Soto, L.; Arciero, D.; Hommes, N.; Whittaker, M.; Arp, D. J. Bacteriol. 2003, 185, 2759-2773. (13) Schmidt, I.; van Spanning, R. J.; Jetten, M. S. Microbiology 2004, 150, 4107-4114. (14) Beaumont, H. J.; van Schooten, B.; Lens, S. I.; Westerhoff, H. V.; van Spanning, R. J. J. Bacteriol. 2004, 186, 4417-4421. (15) Iverson, T. M.; Hendrich, M. P.; Arciero, D. M.; Hooper, A. B.; Rees, D. C. In Handbook of Metalloproteins; Messerschmidt, A., Huber, R., Poulos, T. L., Wieghardt, K., Eds.; John Wiley & Sons: Chichester, 2001, 136146. 10.1021/ja055183+ CCC: $33.50 © 2006 American Chemical Society
NO Reductase Activity from Tetraheme Cytochrome c554
(hemes 2/4, hemes 1/3). Similar heme packing motifs have been observed in a family multiheme proteins, which includes hydroxylamine oxidoreductase (HAO),18 cytochrome c nitrite reductase (NiR),19,20 fumarate reductase,21-23 NapB,24 diheme cytochrome c,25 and the split-Soret cytochrome.26 Only HAO and NiR have significant conservation of primary sequence.27 Cyt c554 can act as the physiological electron acceptor of the enzyme HAO.28-30 Electrons from cyt c554 are thought to pass through a membrane-anchored tetraheme cytochrome cM552 to ubiquinone and then to ammonia monooxygenase (AMO) and the respiratory pathway leading to ATP synthesis.31 In keeping with key roles in the energy-yielding reactions of Nitrosomonas, the cellular concentrations of AMO, HAO, cyt c554, and cytochrome aa3 are within the same order of magnitude.30 The structural arrangement of heme 2 and heme 4 in cyt c554 aligns with that of the active site heme pairs in the enzymes HAO and NiR (each containing one 5-coordinate heme). However, the structure of oxidized and reduced cyt c554 show that, unlike HAO and NiR, the sixth coordination site of the five coordinate heme 2 in cyt c554 is tightly shielded by three hydrophobic amino acid residues (T154, P155, F156). Consistent with this aspect of the structure, cyt c554 does not bind the common heme ligands CN- and CO.32 Therefore, while a possible enzymatic role for cyt c554 in ammonia metabolism has been speculated, until now, there was no evidence in support. We demonstrate here the formation and characterization of a NO bound complex with heme 2 of cyt c554. The presence of NO is shown to result in fast oxidation of the fully ferrous protein, and in multiple turnover reactions, the NO oxidation of the protein is observed to be catalytic. The kinetics of NO reduction by cyt c554, and intermolecular electron transfer in cyt c554 have been measured. Thus, Cyt c554 may account for the some of the unidentified NO reductase activity in Nitrosomonas. Materials and Methods Purification of the Protein. Growth and 57Fe enrichment of Nitrosomonas europaea and purification of cyt c554 and HAO were as (16) Iverson, T. M.; Arciero, D. M.; Hsu, B. T.; Logan, M. S. P.; Hooper, A. B.; Rees, D. C. Nat. Struct. Biol. 1998, 5, 1005-1012. (17) Iverson, T. M.; Arciero, D. M.; Hooper, A. B.; Rees, D. C. J. Biol. Inorg. Chem. 2001, 6, 390-397. (18) Igarashi, N.; Moriyama, H.; Fujiwara, T.; Fukumori, Y.; Tanaka, N, Nat. Struct. Biol. 1997, 4, 276-284. (19) Einsle, O.; Messerschmidt, A.; Stach, P.; Bourenkov, G. P.; Bartunik, H. D.; Huber, R.; Kroneck, P. M. H. Nature 1999, 400, 476-480. (20) Bamford, V. A.; Angove, H. C.; Seward, H. E.; Thomson, A. J.; Cole, J. A.; Butt, J. N.; Hemmings, A. M.; Richardson, D. J. Biochemistry 2002, 41, 2921-2931. (21) Bamford, V.; Dobbin, P. S.; Richardson, D. J.; Hemmings, A. M. Nat. Struct. Biol. 1999, 6, 1104-1107. (22) Taylor, P.; Pealing, S. L.; Reid, G. A.; Chapman, S. K.; Walkinshaw, M. D. Nat. Struct. Biol. 1999, 6, 1108-1112. (23) Leys, D.; Tsapin, A. S.; Nealson, K. H.; Meyer, T. E.; Cusanovich, M. A.; Van Beeumen, J. J. Nat. Struct. Biol. 1999, 6, 1113-1117. (24) Brige, A.; Leys, D.; Meyer, T. E.; Cusanovich, M. A.; Van Beeumen, J. J. Biochemistry 2002, 41, 4827-4836. (25) Heitmann, D.; Einsle, O. Biochemistry 2005, 44, 12411-12419. (26) Matias, P. M.; Morais, J.; Coelho, A. V.; Meijers, R.; Gonzalez, A.; Thompson, A. W.; Sieker, L.; Legall, J.; Carrondo, M. A. J. Biol. Inorg. Chem. 1997, 2, 507-514. (27) Bergmann, D. J.; Hooper, A. B.; Klotz, M. G. Appl. EnViron. Microbiol. 2005, 71, 5371-5382. (28) Yamanaka, T.; Shinra, M. J. Biochem. (Tokyo) 1974, 75, 1265-1273. (29) Arciero, D. M.; Balny, C.; Hooper, A. B. Biochemistry 1991, 30, 1146611472. (30) DiSpirito, A. A.; Taaffe, L. R.; Hooper, A. B. Biochim. Biophys. Acta 1985, 806, 320-330. (31) Whittaker, M.; Bergmann, D.; Arciero, D.; Hooper, A. B. Biochim. Biophys. Acta 2000, 1459, 346-355. (32) Upadhyay, A. K.; Petasis, D. T.; Arciero, D. M.; Hooper, A. B.; Hendrich, M. P. J. Am. Chem. Soc. 2003, 125, 1738-1747.
ARTICLES described previously.32,33 All experiments were carried out in 50 mM potassium phosphate buffer pH 7.0, unless otherwise noted. The concentration of cyt c554 was determined spectrophotometrically with 554 ) 24.6 mM-1 cm-1 per heme.34 All chemicals were reagent grade or better. Doubly distilled or Millipore super Q water was used throughout. Preparation of the Reduced Samples. HAO-mediated reduction of cyt c554 by hydroxylamine (hydroxylamine/HAO reduced cyt c554) or hydrazine (hydrazine/HAO reduced cyt c554) was accomplished with 5-fold excess of substrate (Sigma) in the presence of a catalytic amount (5% molar ratio) of HAO. The protein solutions were incubated for 10 min at room temperature before freezing in liquid nitrogen. The electrochemically half-reduced samples (-50 mV poised) were prepared as reported previously.32 Reduction with sodium dithionite (Sigma) was done with thoroughly degassed protein samples under argon by adding the required amount of sodium dithionite. The sodium dithionite solution was standardized with methyl viologen (570 ) 9000 M-1 cm-1). Photoreduced samples were prepared under argon with 10% riboflavin (vs protein concentration) and 5-fold excess of EDTA by illuminating the sample for 2 h with a projector lamp.35 During illumination, the sample holder was in a chilled water bath in a Pyrex beaker. Preparation of NO Treated Samples. NO gas (Matheson) was passed through water, followed by a solid KOH column to remove trace amounts of acidic N-oxide impurities. The samples were degassed thoroughly in a Schlenk line under argon in septa sealed vials. Measured volumes of NO gas were then bubbled through the degassed protein solutions, using a gastight syringe (Hamilton), followed by inversion of the vial a few times to mix. The protein sample was then transferred, with a gastight syringe, to an EPR tube or Mo¨ssbauer cup under argon, and frozen in liquid nitrogen within five minutes of NO addition. For optical experiments, the protein samples (50 mM KPi, pH 7.0) were degassed in septa sealed cuvettes using a Schlenk line under argon, and reduced with sodium dithionite. A measured volume of NO gas was then bubbled through the cuvette using a gastight syringe, and spectra were recorded immediately. Preparation of the CO Treated Samples. CO gas (Sigma) was passed through degassed water and then bubbled through the protein solutions for 20 min. To establish that the solution contained CO, a myoglobin (Sigma) solution was prepared under similar conditions and checked optically for binding of CO. Kinetic Measurement of NO Reduction. The kinetic parameters for single turnover were determined by monitoring the rate of oxidation of a fully reduced cyt c554 sample in the presence of NO. A 1.85 µM sample of cyt c554 in an optical cell was titrated with dithionite to the fully reduced state with minimal dithionite in solution. The reaction was initiated with by injection of 100 µL of NO saturated solution (∼2 mM at 20 °C)36 to give a final concentration of approximately 125 µM NO. All solutions were degassed thoroughly under argon. EPR and Mo1 ssbauer Spectroscopy. X-band EPR spectra were recorded on a Bruker 300 spectrometer equipped with an Oxford ESR910 liquid helium cryostat and a Bruker bimodal cavity. The quantification of all signals was relative to a CuEDTA spin standard. The spectra were obtained with a field modulation of 1 mTpp at 100 kHz. The magnetic field was calibrated with an NMR gaussmeter and the microwave frequency was measured with a counter. The Q-band spectra were recorded on a Bruker 200 spectrometer with a home-built microwave probe and cryostat.37 Mo¨ssbauer spectra were obtained on a constant acceleration instrument and isomer shifts are reported with respect to an iron metal standard. All displayed spectra were recorded on 57Fe enriched protein. (33) Andersson, K. K.; Kent, T. A.; Lipscomb, J. D.; Hooper, A. B.; Munck, E. J. Biol. Chem. 1984, 259, 6833-6840. (34) Andersson, K. K.; Lipscomb, J. D.; Valentine, M.; Munck, E.; Hooper, A. B. J. Biol. Chem. 1986, 261, 1126-1138. (35) Massey, V.; Stankovich, M.; Hemmerich, P. Biochemistry 1978, 17, 1-8. (36) Wilhelm, E.; Battino, R.; Wilcock, R. J. Chem. ReV. 1977, 77, 219-262. (37) Petasis, D. T.; Hendrich, M. P. J. Magn. Reson. 1999, 136, 200-206. J. AM. CHEM. SOC.
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ARTICLES
Figure 1. Mo¨ssbauer spectra of (A) hydrazine/HAO reduced and (B) hydroxylamine/HAO reduced 1.7 mM cyt c554, temperature 4.2 K, in absence of an applied magnetic field. The brackets labeled IR and IIR mark the doublets arising from ferrous low-spin heme 1 and ferrous high-spin heme 2. The spectrum in (C) is the difference B-A. The solid lines are fits (see text for parameters), all using line widths of 0.32 mm/s.
The EPR simulation software was written by the authors. The program diagonalizes the spin Hamiltonian
Hs ) JS1‚S2 + S1‚D′‚S2 + dS1 × S2 + βB‚g1‚S1 + βB‚g2‚S2
(1)
where J is the isotropic exchange coupling between two heme centers, D′ is a symmetric traceless tensor, d is a polar vector of the antisymmetric contribution, and g1, g2 are the intrinsic g-tensors of two interacting hemes. The simulations are generated with consideration of all intensity factors, which allows computation of simulated spectra for a specified sample concentration. The simulations therefore allow a quantitative determination of protein signal intensities. The Windows software package (SpinCount) is available for general application to any mono- or dinuclear metal complex by contacting Prof. Hendrich.
Results
Mo1 ssbauer Spectroscopy. We have shown previously that two of the hemes (heme 1 and 2) of cyt c554 can be reduced with the substrates of HAO (hydrazine or hydroxylamine) in the presence of catalytic amounts (5%) of HAO.32 Cyt c554 does not react directly with these substrates. As mentioned briefly in our previous work, the spectroscopic properties of cyt c554 reduced by HAO in the presence of NH2OH as opposed to N2H4 are quite different, and are not attributable to an altered redox state. Low temperature (4.2 K) Mo¨ssbauer spectra, in the absence of an applied magnetic field, of the hydrazine/HAO and hydroxylamine/HAO reduced samples are shown in Figure 1A and B, respectively. The hydrazine reduced spectrum shows paramagnetic hyperfine features from the two low-spin (S ) 1/2) ferric hemes (hemes 3 and 4), and two diamagnetic doublets from high-spin (S ) 2) ferrous heme 2 (IIR) and low-spin (S ) 0) ferrous heme 1 (IR). The solid line overlaid on the spectrum is a fit to the diamagnetic ferrous doublets of heme 1 (IR) and heme 2 (IIR) with δ ) 0.42 mm/s, ∆EQ ) 1.3 mm/s and δ ) 0.98 mm/s, ∆EQ ) 2.2 mm/s, respectively, each accounting for 4332 J. AM. CHEM. SOC.
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25% of the total iron. The hydroxylamine/HAO reduced spectrum (Figure 1B) shows paramagnetic hyperfine features similar to the hydrazine/HAO spectrum, however, the intensity of the diamagnetic doublets has changed. The intensity of the high-spin ferrous doublet (IIR) has dropped to 50% of the hydrazine/HAO reduced sample, with the appearance of a new diamagnetic doublet (X). The new doublet X is apparent in the difference spectrum hydroxylamine/HAO minus hydrazine/HAO (Figure 1C). The theoretical curve overlaid on the difference spectrum is a fit with δ ) 0.23 mm/s, ∆EQ ) 1.53 mm/s, accounting for 12% of the total iron. The positive peaks in the subtraction are due to the decrease in the intensity of the doublet associated with the high-spin ferrous heme. The solid line overlaid in Figure 1B is a theoretical curve which shows the effect of loss of 50% of the high-spin heme and the addition of species X. The Mossbauer parameters of species X are close to those previously reported for ferrous heme-NO [FeNO]7 protein species,38-41 and differ significantly from those of known ferric heme-NO [FeNO]6 protein species.42 Heme [FeNO]7 complexes have spin S ) 1/2 and display paramagnetic Mo¨ssbauer spectra, but species X we observe displays a quadrupole doublet. As we shall demonstrate from the EPR data below, the (heme 2)NO [FeNO]7 complex is spin-coupled to low-spin (S ) 1/2) ferric heme 4. The (heme 2)NO complex is S ) 1/2, but the spin-coupling to heme 4 results in fast spin relaxation and low spin expectation, and thus the spectrum shows a quadrupole doublet. To test for binding of NO or CO to the high-spin heme 2 center, Mo¨ssbauer samples of fully reduced cyt c554 were treated with excess of NO or CO gas. The high temperature (150 K) Mo¨ssbauer spectrum (not shown) of reduced cyt c554 prior to addition was the same as reported previously.34 Upon addition of NO, the spectrum showed a minor (10%) drop in the intensity of the high-spin ferrous doublet (IIR) without a significant change in the other doublets. Upon addition of CO, the spectrum showed no change in any of the doublets. Thus, NO and CO did not appear to bind in significant amount to the fully reduced protein. However, as we will show below, NO does specifically react with the high-spin ferrous heme 2 center in the fully reduced state of the protein, forming an transient NO bound species that is detectable under limiting reductant conditions. EPR Spectroscopy of Substrate/HAO Reduced cyt c554. X- and Q-band EPR spectra of substrate/HAO reduced cyt c554 samples are shown in Figure 2 and Figure 3. For both figures, the experimental spectra (solid lines) of the hydrazine/HAO reduced samples are shown in A, and hydroxylamine/HAO reduced samples in C, and the dashed lines are quantitative simulations. The sharp signal near g ) 2.07 (Figure 2A) is from a Cu2+ impurity (
