Cytochrome c nitrite reductase from the bacterium Geobacter lovleyi represents a new NrfA subclass
2020; Elsevier BV; Volume: 295; Issue: 33 Linguagem: Inglês
10.1074/jbc.ra120.013981
ISSN1083-351X
AutoresJulius Campeciño, Satyanarayana Lagishetty, Z. Wawrzak, Victor Sosa Alfaro, Nicolai Lehnert, Gemma Reguera, Jian Hu, Eric L. Hegg,
Tópico(s)Ammonia Synthesis and Nitrogen Reduction
ResumoCytochrome c nitrite reductase (NrfA) catalyzes the reduction of nitrite to ammonium in the dissimilatory nitrate reduction to ammonium (DNRA) pathway, a process that competes with denitrification, conserves nitrogen, and minimizes nutrient loss in soils. The environmental bacterium Geobacter lovleyi has recently been recognized as a key driver of DNRA in nature, but its enzymatic pathway is still uncharacterized. To address this limitation, here we overexpressed, purified, and characterized G. lovleyi NrfA. We observed that the enzyme crystallizes as a dimer but remains monomeric in solution. Importantly, its crystal structure at 2.55-Å resolution revealed the presence of an arginine residue in the region otherwise occupied by calcium in canonical NrfA enzymes. The presence of EDTA did not affect the activity of G. lovleyi NrfA, and site-directed mutagenesis of this arginine reduced enzymatic activity to <3% of the WT levels. Phylogenetic analysis revealed four separate emergences of Arg-containing NrfA enzymes. Thus, the Ca2+-independent, Arg-containing NrfA from G. lovleyi represents a new subclass of cytochrome c nitrite reductase. Most genera from the exclusive clades of Arg-containing NrfA proteins are also represented in clades containing Ca2+-dependent enzymes, suggesting convergent evolution. Cytochrome c nitrite reductase (NrfA) catalyzes the reduction of nitrite to ammonium in the dissimilatory nitrate reduction to ammonium (DNRA) pathway, a process that competes with denitrification, conserves nitrogen, and minimizes nutrient loss in soils. The environmental bacterium Geobacter lovleyi has recently been recognized as a key driver of DNRA in nature, but its enzymatic pathway is still uncharacterized. To address this limitation, here we overexpressed, purified, and characterized G. lovleyi NrfA. We observed that the enzyme crystallizes as a dimer but remains monomeric in solution. Importantly, its crystal structure at 2.55-Å resolution revealed the presence of an arginine residue in the region otherwise occupied by calcium in canonical NrfA enzymes. The presence of EDTA did not affect the activity of G. lovleyi NrfA, and site-directed mutagenesis of this arginine reduced enzymatic activity to <3% of the WT levels. Phylogenetic analysis revealed four separate emergences of Arg-containing NrfA enzymes. Thus, the Ca2+-independent, Arg-containing NrfA from G. lovleyi represents a new subclass of cytochrome c nitrite reductase. Most genera from the exclusive clades of Arg-containing NrfA proteins are also represented in clades containing Ca2+-dependent enzymes, suggesting convergent evolution. Correction: Cytochrome c nitrite reductase from the bacterium Geobacter lovleyi represents a new NrfA subclassJournal of Biological ChemistryVol. 298Issue 12PreviewThe authors report that in Figure S4 the value 1.63 of NrfA Ve/V0 is incorrect due to a typo and that the actual value of NrfA Ve/V0 is 1.93. Using this the corrected Ve/V0 value of 1.93 results in a calculated NrfA molecular weight of approximately 52 kDa. This new calculated value agrees well with the actual molecular weight of NrfA (∼56 kDa), and it further supports the conclusion that NrfA behaves as a monomer in solution under these conditions. Full-Text PDF Open Access The global nitrogen cycle is an indispensable biogeochemical process, as nitrogen is a fundamental component of every living organism (1Lehnert N. Dong H.T. Harland J.B. Hunt A.P. White C.J. Reversing nitrogen fixation.Nat. Rev. Chem. 2018; 2: 278-28910.1038/s41570-018-0041-7Crossref Scopus (92) Google Scholar). 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The relevance of the two calcium sites in the structure of the catalytic subunit (NrfA).J. Biol. Chem. 2003; 278 (12618432): 17455-1746510.1074/jbc.M211777200Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar), the enzyme is a dimer and contains five heme c cofactors per monomer with a Ca2+ ion located at the distal side of the heme-1 active site. Although the exact role of the calcium ion is uncertain, it is proposed to be involved in proton delivery (14Bykov D. Neese F. Six-electron reduction of nitrite to ammonia by cytochrome c nitrite reductase: insights from density functional theory studies.Inorg. Chem. 2015; 54 (26237518): 9303-931610.1021/acs.inorgchem.5b01506Crossref PubMed Scopus (55) Google Scholar) and structural stability (13Cunha C.A. Macieira S. Dias J.M. Almeida G. Goncalves L.L. Costa C. Lampreia J. Huber R. Moura J.J. Moura I. Romão M.J. Cytochrome c nitrite reductase from Desulfovibrio desulfuricans ATCC 27774. 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The metal content was further confirmed by inductively coupled plasma-optical emission spectroscopy (ICP-OES) (Table S1). The UV-Vis and EPR spectra (Fig. S2 and S3) are consistent with previously characterized NrfA enzymes (10Bamford V.A. Angove H.C. Seward H.E. Thomson A.J. Cole J.A. Butt J.N. Hemmings A.M. Richardson D.J. Structure and spectroscopy of the periplasmic cytochrome c nitrite reductase from Escherichia coli.Biochemistry. 2002; 41 (11863430): 2921-293110.1021/bi015765dCrossref PubMed Scopus (134) Google Scholar, 22Ali M. Stein N. Mao Y. Shahid S. Schmidt M. Bennett B. Pacheco A.A. Trapping of a putative intermediate in the cytochrome c nitrite reductase (ccNiR)-catalyzed reduction of nitrite: implications for the ccNiR reaction mechanism.J. Am. Chem. Soc. 2019; 141 (31381304): 13358-1337110.1021/jacs.9b03036Crossref PubMed Scopus (10) Google Scholar, 23Pereira I.A.C. Legall J. Xavier A.V. Teixeira M. 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A complicating factor with this approach, however, is the dilution that naturally occurs during chromatography, making it challenging to assess the dependence of the oligomerization state on enzyme concentration. To bypass this limitation, we used dynamic light scattering (DLS) to measure the hydrodynamic radius (RH) of the enzyme in solution at the concentration of interest (29Nobbmann U. Connah M. Fish B. Varley P. Gee C. Mulot S. Chen J. Zhou L. Lu Y. Shen F. Yi J. Harding S.E. Dynamic light scattering as a relative tool for assessing the molecular integrity and stability of monoclonal antibodies.Biotechnol. Genet. Eng. Rev. 2007; 24 (18059629): 117-12810.1080/02648725.2007.10648095Crossref PubMed Scopus (144) Google Scholar, 30Borgstahl G.E. How to use dynamic light scattering to improve the likelihood of growing macromolecular crystals.in: Doublie S. Walker J.M. Methods in Molecular Biology. Humana Press, Totowa, New Jersey2007: 109-129Google Scholar). The hydrodynamic radius measured using this technique reflects the average contribution from each oligomer. For G. lovleyi NrfA, the theoretical monomer and dimer RH is 3.3 nm and 4.5 nm, respectively. These calculated radii are in close agreement with those estimated for globular proteins of similar size (31Stetefeld J. McKenna S.A. Patel T.R. Dynamic light scattering: a practical guide and applications in biomedical sciences.Biophys. Rev. 2016; 8 (28510011): 409-42710.1007/s12551-016-0218-6Crossref PubMed Scopus (808) Google Scholar). The consistency of the radius measured at increasing concentrations (Table 1) indicated that G. lovleyi NrfA remains monomeric in solution even at very high concentrations. This result was consistent with the polydispersity index measured for each sample. A sample containing only a single species is expected to have a polydispersity index of less than 15% (30Borgstahl G.E. How to use dynamic light scattering to improve the likelihood of growing macromolecular crystals.in: Doublie S. Walker J.M. Methods in Molecular Biology. Humana Press, Totowa, New Jersey2007: 109-129Google Scholar).Table 1Data from dynamic light scatteringConcentration (µm)RH (nm)Polydispersity index (%)243.411.9523.610.91053.411.91653.411.91753.412.52663.313.63273.14.3 Open table in a new tab Although G. lovleyi NrfA is a monomer in solution, it crystallized as a dimer, consistent with other structurally characterized NrfA homologs. Alignment of the G. lovleyi NrfA crystal structure with that from Sulfurospirillum deleyianum (PDB entry 1QDB), its closest structurally characterized homolog (Fig. S5), revealed a C-α root mean square deviation (RMSD) of 2.67 (PyMOL, Schrödinger, Inc.). The crystal, which resolved at 2.55 Å, belongs to the primitive monoclinic space group P212121 with unit cell a = 110.9, b = 144.6, and c = 234.9 (Table 2). The dimer interface is dominated by three α-helices from each monomer, and it is primarily stabilized by a strong electrostatic contribution from two salt bridges (Fig. 1A) as well as H-bonding interactions. The structure revealed four bis-His coordinated hemes as well as a Lys-ligated heme (heme-1) that constitutes the active site. The hemes from this structure aligned well with the hemes from S. deleyianum NrfA (Fig. 1B). The substrate binding site at the distal side of the active-site heme-1 is occupied by sulfate (efforts to obtain high-quality crystals with nitrite bound were unsuccessful), which is stabilized by the active-site residues Tyr-221, His-278, and Arg-122, as well as a water molecule (Fig. 2A). These active-site residues are strictly conserved in all NrfA enzymes characterized to date.Table 2Crystallographic statisticsParameterValue(s) for G. lovleyi NrfA-1Data collection BeamlineLS-CAT (21-ID-D) Wavelength (Å)1.72 Space groupP212121 Unit cell a, b, c (Å)110.9 144.6 234.9 α, β, γ (∘)90.0, 90.0, 90.0 ResolutionaHighest resolution shell is shown in parentheses. (Å)29.36–2.55 (2.64–2.55) RedundancyaHighest resolution shell is shown in parentheses.5.5 (5.3) CompletenessaHighest resolution shell is shown in parentheses. (%)98.9 (98.8) I/σIaHighest resolution shell is shown in parentheses.4.8 (1.8) RmergeaHighest resolution shell is shown in parentheses.,bRmerge=∑hkl∑j|Ij(hkl)− |/∑hkl∑jIj(hkl), where I is the intensity of reflection.0.20 (1.66) RpimaHighest resolution shell is shown in parentheses.,cRpim=∑hkl[1/(N−1)]1/2∑j|Ij(hkl)− |/∑hkl∑jIj/(hkl), where N is the redundancy of the dataset.0.11 (0.77) CC1/20.36dAverage CC1/2 was not reported by the version of HKL2000 used in this work.Refinement No. of unique reflections122,238 No. of atoms23,376 Protein (n)21,161 Heme (n)1290 Sulfate (n)30 Rwork/RfreeeRwork=∑hkl||Fobs|−|Fcalc||/∑hkl|Fobs|, where Fobs and Fcalc is the observed and the calculated structure factor, respectively. Rfree is the cross-validation R factor for the test set of reflections (5% of the total) omitted from model refinement.0.19/0.24 Wilson B-factor (Å2)34.2 B-factor (Å2) Protein39.7 Heme31.6 Sulfate35.7 Solvent33.9 RMSD Bond length (Å)0.010 Bond angle (∘)1.21 Ramachandran plot (%) Favored96.0 Allowed3.5 Outliers0.5a Highest resolution shell is shown in parentheses.b Rmerge=∑hkl∑j|Ij(hkl)− |/∑hkl∑jIj(hkl), where I is the intensity of reflection.c Rpim=∑hkl[1/(N−1)]1/2∑j|Ij(hkl)− |/∑hkl∑jIj/(hkl), where N is the redundancy of the dataset.d Average CC1/2 was not reported by the version of HKL2000 used in this work.e Rwork=∑hkl||Fobs|−|Fcalc||/∑hkl|Fobs|, where Fobs and Fcalc is the observed and the calculated structure factor, respectively. Rfree is the cross-validation R factor for the test set of reflections (5% of the total) omitted from model refinement. Open table in a new tab Figure 2G. lovleyi (A) and S. deleyianum (PDB entry ) (B) NrfA active sites. A red sphere represents a water molecule, and a yellow dash represents either an H-bond or a salt bridge. The G. lovleyi NrfA active-site difference density map is provided in Fig. S6.View Large Image Figure ViewerDownload Hi-res image Download (PPT) A key structural difference between G. lovleyi NrfA and the previously crystallized homologs can be found at the active-site region. Specifically, no calcium was found near the heme-1 active site in G. lovleyi NrfA. Instead, the active site contains an arginine residue (Arg-277) in the region that would otherwise be occupied by a calcium ion in other NrfA homologs (Fig. 2). Arg-277 forms a salt bridge with Glu-263 as well as H-bonds to both the side chain and amide oxygen atoms of the active-site tyrosine (Tyr-221). In addition, there is a π-cation interaction between Arg-277 and Tyr-221. In the Ca2+-containing homologs, the amide oxygen of this same tyrosine is ligated to the calcium (Fig. 2B). In contrast to the presence of two well-ordered H2O molecules coordinated to the calcium ion in the Ca2+-containing homologs, the presence of a water molecule in the vicinity of Arg-277 in G. lovleyi NrfA could not be definitively ascertained (i.e. only two out of six monomers revealed a well-ordered water molecule H-bonded to Arg-277). Interestingly, the positive and negative charges at both the Ca2+ site and the Arg site are more or less balanced. To achieve this, the calcium is coordinated by Glu-216, its two H2O ligands are H-bonded to Asp-267 (Fig. 2B), and its lysine ligand is H-bonded to Asp-400 (not shown). In G. lovleyi NrfA, however, charge balance is achieved by Arg-277 forming a salt bridge to Glu-263 (Fig. 2A). It is important to note that except for Desulfovibrio desulfuricans, all NrfA enzymes crystallographically characterized thus far, including G. lovleyi NrfA, were purified and crystallized without the addition of calcium to any of the buffers (8Einsle O. Messerschmidt A. Stach P. Bourenkov G.P. Bartunik H.D. Huber R. Kroneck P.M. Structure of cytochrome c nitrite reductase.Nature. 1999; 400 (10440380): 476-48010.1038/22802Crossref PubMed Scopus (271) Google Scholar, 9Einsle O. Stach P. Messerschmidt A. Simon J. Kröger A. Huber R. Kroneck P.M. Cytochrome c nitrite reductase from Wolinella succinogenes: structure at 1.6 Å resolution, inhibitor binding, and heme-packing motifs.J. Biol. Chem. 2000; 275 (10984487): 39608-3961610.1074/jbc.M006188200Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar, 10Bamford V.A. Angove H.C. Seward H.E. Thomson A.J. Cole J.A. Butt J.N. Hemmings A.M. Richardson D.J. Structure and spectroscopy of the periplasmic cytochrome c nitrite reductase from Escherichia coli.Biochemistry. 2002; 41 (11863430): 2921-293110.1021/bi015765dCrossref PubMed Scopus (134) Google Scholar, 11Youngblut M. Judd E.T. Srajer V. Sayyed B. Goelzer T. Elliott S.J. Schmidt M. Pacheco A.A. Laue crystal structure of Shewanella oneidensis cytochrome c nitrite reductase from a high-yield expression system.J. Biol. Inorg. Chem. 2012; 17 (22382353): 647-66210.1007/s00775-012-0885-0Crossref PubMed Scopus (44) Google Scholar, 12Rodrigues M.L. Oliveira T.F. Pereira I.A. Archer M. X-ray structure of the membrane-bound cytochrome c quinol dehydrogenase NrfH reveals novel haem coordination.EMBO J. 2006; 25 (17139260): 5951-596010.1038/sj.emboj.7601439Crossref PubMed Scopus (130) Google Scholar, 13Cunha C.A. Macieira S. Dias J.M. Almeida G. Goncalves L.L. Costa C. Lampreia J. Huber R. Moura J.J. Moura I. Romão M.J. Cytochrome c nitrite reductase from Desulfovibrio desulfuricans ATCC 27774. The relevance of the two calcium sites in the structure of the catalytic subunit (NrfA).J. Biol. Chem. 2003; 278 (12618432): 17455-1746510.1074/jbc.M211777200Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar). In addition, our attempts to crystallize G. lovleyi NrfA in the presence of calcium yielded poorly diffracting crystals. Using CAVER software (32Chovancova E. Pavelka A. Benes P. Strnad O. Brezovsky J. Kozlikova B. Gora A. Sustr V. Klvana M. Medek P. Biedermannova L. Sochor J. Damborsky J. CAVER 3.0: a tool for the analysis of transport pathways in dynamic protein structures.PLoS Comput. Biol. 2012; 8: 1-1210.1371/journal.pcbi.1002708Crossref PubMed Scopus (748) Google Scholar), we identified a tunnel connecting the putative substrate and product channels (Fig. 3). The substrate channel in G. lovleyi NrfA is in a location similar to that in the S. deleyianum NrfA protein (8Einsle O. Messerschmidt A. Stach P. Bourenkov G.P. Bartunik H.D. Huber R. Kroneck P.M. Structure of cytochrome c nitrite reductase.Nature. 1999; 400 (10440380): 476-48010.1038/22802Crossref PubMed Scopus (271) Google Scholar), and it is highly cationic because of the presence of several positively charged amino acid residues in the vicinity, i.e. Lys-124, Lys-225, Arg-277, and Arg-122. Whereas Arg-122 is strictly conserved in all NrfA proteins and Arg-277 is strictly conserved in Arg-containing NrfA proteins, Lys-124 and Lys-225 are highly conserved in Arg-containing but not in Ca2+-containing NrfA homologs. Conversely, the product channel in G. lovleyi NrfA diverges from that of S. deleyianum. In S. deleyianum, the product channel hovers above heme-4 while in G. lovleyi it is near heme-3. Furthermore, the G. lovleyi NrfA product channel is surrounded with negatively charged carboxylates from heme-1 and heme-3 as well as from amino acid residues Glu-101 and Asp-114. These residues guide the exit of the positively charged ammonium ion. In NrfA assays, dithionite-reduced methyl viologen was used as an artificial electron donor to drive the enzymatic reduction of nitrite. The disappearance of the intense blue color from the reduced methyl viologen was monitored to measure the activity of the enzyme. Despite lacking the calcium ion, G. lovleyi NrfA demonstrates kinetic behavior similar to that of previously characterized homologs. It displays zero-order kinetics with respect to the concentration of methyl viologen, first-order kinetics with respect to the concentration of the enzyme, and hyperbolic dependence of the rate with respect to the concentration of nitrite (Fig. 4). The KM and the kcat values obtained from the Michaelis-Menten curve were 27 ± 2 μm NO2− and 1,291 ± 34 μmol NO2− min−1 mg−1 enzyme, respectively. The G. lovleyi NrfA KM is similar to that reported for NrfA proteins purified from E. coli (10Bamford V.A. Angove H.C. Seward H.E. Thomson A.J. Cole J.A. Butt J.N. Hemmings A.M. Richardson D.J. Structure and spectroscopy of the periplasmic cytochrome c nitrite reductase from Escherichia coli.Biochemistry. 2002; 41 (11863430): 2921-293110.1021/bi015765dCrossref PubMed Scopus (134) Google Scholar) and S. oneidensis (11Youngblut M. Judd E.T. Srajer V. Sayyed B. Goelzer T. Elliott S.J. Schmidt M. Pacheco A.A. Laue crystal structure of Shewanella oneidensis cytochrome c nitrite reductase from a high-yield expression system.J. Biol. Inorg. Chem. 2012; 17 (22382353): 647-66210.1007/s00775-012-0885-0Crossref PubMed Scopus (44) Google Scholar). Whereas the kca
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