Nitric Oxide Binding to Prokaryotic Homologs of the Soluble Guanylate Cyclase β1 H-NOX Domain
2006; Elsevier BV; Volume: 281; Issue: 31 Linguagem: Inglês
10.1074/jbc.m600557200
ISSN1083-351X
AutoresElizabeth M. Boon, Joseph H. Davis, Rosalie Tran, David S. Karow, Shirley Huang, Duohai Pan, Michael M. Miazgowicz, Richard A. Mathies, Michael A. Marletta,
Tópico(s)Nitric Oxide and Endothelin Effects
ResumoThe heme cofactor in soluble guanylate cyclase (sGC) is a selective receptor for NO, an important signaling molecule in eukaryotes. The sGC heme domain has been localized to the N-terminal 194 amino acids of the β1 subunit of sGC and is a member of a family of conserved hemoproteins, called the H-NOX family (Heme-Nitric Oxide and/or OXygen-binding domain). Three new members of this family have now been cloned and characterized, two proteins from Legionella pneumophila (L1 H-NOX and L2 H-NOX) and one from Nostoc punctiforme (Np H-NOX). Like sGC, L1 H-NOX forms a 5-coordinate FeII-NO complex. However, both L2 H-NOX and Np H-NOX form temperature-dependent mixtures of 5- and 6-coordinate FeII-NO complexes; at low temperature, they are primarily 6-coordinate, and at high temperature, the equilibrium is shifted toward a 5-coordinate geometry. This equilibrium is fully reversible with temperature in the absence of free NO. This process is analyzed in terms of a thermally labile proximal FeII-His bond and suggests that in both the 5- and 6-coordinate FeII-NO complexes of L2 H-NOX and Np H-NOX, NO is bound in the distal heme pocket of the H-NOX fold. NO dissociation kinetics for L1 H-NOX and L2 H-NOX have been determined and support a model in which NO dissociates from the distal side of the heme in both 5- and 6-coordinate complexes. The heme cofactor in soluble guanylate cyclase (sGC) is a selective receptor for NO, an important signaling molecule in eukaryotes. The sGC heme domain has been localized to the N-terminal 194 amino acids of the β1 subunit of sGC and is a member of a family of conserved hemoproteins, called the H-NOX family (Heme-Nitric Oxide and/or OXygen-binding domain). Three new members of this family have now been cloned and characterized, two proteins from Legionella pneumophila (L1 H-NOX and L2 H-NOX) and one from Nostoc punctiforme (Np H-NOX). Like sGC, L1 H-NOX forms a 5-coordinate FeII-NO complex. However, both L2 H-NOX and Np H-NOX form temperature-dependent mixtures of 5- and 6-coordinate FeII-NO complexes; at low temperature, they are primarily 6-coordinate, and at high temperature, the equilibrium is shifted toward a 5-coordinate geometry. This equilibrium is fully reversible with temperature in the absence of free NO. This process is analyzed in terms of a thermally labile proximal FeII-His bond and suggests that in both the 5- and 6-coordinate FeII-NO complexes of L2 H-NOX and Np H-NOX, NO is bound in the distal heme pocket of the H-NOX fold. NO dissociation kinetics for L1 H-NOX and L2 H-NOX have been determined and support a model in which NO dissociates from the distal side of the heme in both 5- and 6-coordinate complexes. The H-NOX (Heme-Nitric Oxide and/or OXygen-binding domain) family of heme proteins has been identified recently, first through sequence analysis (1Iyer L.M. Anantharaman V. Aravind L. BMC Genomics. 2003; 4: 5-12Crossref PubMed Scopus (154) Google Scholar) and then through biochemical characterization (2Karow D.S. Pan D. Tran R. Pellicena P. Presley A. Mathies R.A. Marletta M.A. Biochemistry. 2004; 43: 10203-10211Crossref PubMed Scopus (161) Google Scholar). This family was identified based on homology to the heme domain from soluble guanylate cyclase (sGC), 3The abbreviations used are: sGC, soluble guanylate cyclase; CO, carbon monoxide; NO, nitric oxide; O2, oxygen; ORF, open reading frame; Na2S2O4, dithionite; Mb, myoglobin; Ax cyt c′, cytochrome c′ from Alcaligenes xylosoxidans; Rm FixLN, heme domain of FixL from Rhizogium meliloti; CN, coordination state; HPLC, high pressure liquid chromatography; Tt H-NOX, T. tengcongensis H-NOX.3The abbreviations used are: sGC, soluble guanylate cyclase; CO, carbon monoxide; NO, nitric oxide; O2, oxygen; ORF, open reading frame; Na2S2O4, dithionite; Mb, myoglobin; Ax cyt c′, cytochrome c′ from Alcaligenes xylosoxidans; Rm FixLN, heme domain of FixL from Rhizogium meliloti; CN, coordination state; HPLC, high pressure liquid chromatography; Tt H-NOX, T. tengcongensis H-NOX. the well characterized and conserved eukaryotic nitric oxide receptor (3Denninger J.W. Marletta M.A. Acta Biochem. Biophys. 1999; 1411: 334-350Crossref PubMed Scopus (863) Google Scholar, 4Boon E.M. Marletta M.A. J. Inorg. Biochem. 2005; 99: 892-902Crossref PubMed Scopus (99) Google Scholar). H-NOX proteins have now been identified in many prokaryotes in addition to the well known eukaryotic sGCs. Several of these prokaryotic H-NOX proteins have now been cloned, expressed, and spectroscopically characterized (2Karow D.S. Pan D. Tran R. Pellicena P. Presley A. Mathies R.A. Marletta M.A. Biochemistry. 2004; 43: 10203-10211Crossref PubMed Scopus (161) Google Scholar, 5Boon E.M. Huang S.H. Marletta M.A. Nat. Chem. Biol. 2005; 1: 53-59Crossref PubMed Scopus (167) Google Scholar, 6Karow D.S. Davis J.H. Marletta M.A. Biochemistry. 2005; 44: 16266-16274Crossref PubMed Scopus (67) Google Scholar, 7Nioche P. Berka V. Vipond J. Minton N. Tsai A.L. Raman C.S. Science. 2004; 306: 1550-1553Crossref PubMed Scopus (173) Google Scholar). In addition, the crystal structure of the H-NOX domain from Thermoanaerobacter tengcongensis (Tt H-NOX) has been solved to 1.77 Å resolution (8Pellicena P. Karow D.S. Boon E.M. Marletta M.A. Kuriyan J. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 12854-12859Crossref PubMed Scopus (248) Google Scholar), providing the first structural data for the H-NOX family and, by homology, for the heme domain in the β-subunit of sGC. Taken together, these structural and biochemical results have suggested that some members of this family are able to use a homologous protein fold and an identical heme cofactor to discriminate between NO and O2 binding.Although a great deal of information pertaining to the entire H-NOX family has been determined from studying the structure of Tt H-NOX (5Boon E.M. Huang S.H. Marletta M.A. Nat. Chem. Biol. 2005; 1: 53-59Crossref PubMed Scopus (167) Google Scholar, 8Pellicena P. Karow D.S. Boon E.M. Marletta M.A. Kuriyan J. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 12854-12859Crossref PubMed Scopus (248) Google Scholar), specific structural data for an NO complex to any of the H-NOX proteins are still lacking. This is particularly relevant as NO is an important signaling molecule in eukaryotes, and sGC is the only firmly established NO receptor (3Denninger J.W. Marletta M.A. Acta Biochem. Biophys. 1999; 1411: 334-350Crossref PubMed Scopus (863) Google Scholar). Cytochrome c′ is a protein that shares ligand binding properties but not sequence or structural homology with sGC. A crystal structure of the FeII-NO cytochrome c′ complex shows NO bound to the proximal side of the heme (9Lawson D.M. Stevenson C.E. Andrew C.R. George S.J. Eady R.R. Biochem. Soc. Trans. 2003; 31: 553-557Crossref PubMed Google Scholar), which has led to speculation that the same is true for sGC. Physical studies of NO binding to sGC have been hindered by poor expression systems for this large protein. Thus considerable effort has been spent on shorter constructs of sGC containing its heme-binding domain, such as β1-(1-385) (10Zhao Y. Marletta M.A. Biochemistry. 1997; 36: 15959-15964Crossref PubMed Scopus (107) Google Scholar) and β1-(1-194) (6Karow D.S. Davis J.H. Marletta M.A. Biochemistry. 2005; 44: 16266-16274Crossref PubMed Scopus (67) Google Scholar). Most of the prokaryotic members of the H-NOX family, however, are stand-alone ∼200 amino acid proteins that overexpress well in Escherichia coli. These close homologs of the sGC heme domain have proven generally useful in understanding ligand binding in the family.In this study, we examine three additional prokaryotic H-NOX proteins from Legionella pneumophila (ORF1 and ORF2) and Nostoc punctiforme (Np H-NOX). Unlike all other prokaryotes with predicted H-NOX proteins identified to date, L. pneumophila has two predicted ORFs for H-NOX proteins (L1 H-NOX and L2 H-NOX). Np H-NOX is 39% identical to the H-NOX domain from sGC, and the H-NOX domains L1 H-NOX and L2 H-NOX share 19 and 16% identity with sGC, respectively. The NO binding characteristics of these proteins will be important in determining function in these bacteria. In addition, given the high homology these proteins share with sGC, these results should have implications for the NO-heme complex of sGC.EXPERIMENTAL PROCEDURESMaterials and General Methods—Unless otherwise noted, all reagents were purchased in their highest available purity and used as received.Protein Expression and Purification—PCR was used to amplify ZP_421786 from N. punctiforme genomic DNA (ATCC, Manassas, VA) using Expand polymerase (Roche Applied Science). Upstream and downstream primers contained NdeI and NotI restriction sites, respectively. PCR was used to amplify YP_095089 (L1 H-NOX) and AAU28519 (L2 H-NOX) from L. pneumophila genomic DNA (ATCC) using Expand polymerase (Roche Applied Science). Upstream and downstream primers contained NdeI and XhoI restriction sites, respectively. All amplified PCR products were cloned into pET-20b (Novagen) and sequenced (sequencing core; University of California, Berkeley). Mutagenesis was carried out using the QuikChange® protocol from Stratagene. Cell culture procedures and purification of Np H-NOX were carried out as described previously for the H-NOX protein from Vibrio cholerae (2Karow D.S. Pan D. Tran R. Pellicena P. Presley A. Mathies R.A. Marletta M.A. Biochemistry. 2004; 43: 10203-10211Crossref PubMed Scopus (161) Google Scholar). Cell culture procedures of both Legionella H-NOX proteins were carried out as described previously (2Karow D.S. Pan D. Tran R. Pellicena P. Presley A. Mathies R.A. Marletta M.A. Biochemistry. 2004; 43: 10203-10211Crossref PubMed Scopus (161) Google Scholar). Purification of the Legionella H-NOX proteins took advantage of the C-terminal His6 tag; proteins were purified by metal affinity (nickel-nitrilotriacetic acid) followed by gel filtration (Superdex 200 HiLoad 26/60).Sample Preparation—Preparation of the various protein complexes was carried out as published previously (2Karow D.S. Pan D. Tran R. Pellicena P. Presley A. Mathies R.A. Marletta M.A. Biochemistry. 2004; 43: 10203-10211Crossref PubMed Scopus (161) Google Scholar) with one minor exception, as follows. Rather than generate the FeII-NO complex with NO gas generated from the head space of a concentrated diethylamine NONOate (Cayman) solution, diethylamine NONOate was added directly to the protein solution, and then excess NO, diethylamine, and diethylamine NONOate were removed from the sample by using a PD10 desalting column. This was to ensure no free NO was in solution, and thus the only NO present in the sample was that bound to the heme in the protein.Spectroscopy—All electronic spectra were recorded on a Cary 3E spectrophotometer equipped with a Neslab RTE-100 constant temperature bath. For temperatures at 0 °C or lower, ethylene glycol (50%) was added to the constant temperature bath and 5% glycerol to the protein sample. Resonance Raman spectra were collected using 406.7 nm excitation from a Kr+ laser (Spectra-Physics model 2025). Raman scattering was detected with a cooled, back-illuminated CCD (LN/CCD-1100/PB; Roper Scientific) controlled by an ST-133 controller coupled to a subtractive dispersive double spectrograph. The laser power at the sample was ∼2 milliwatts. A microspinning sample cell was used to minimize photo-induced degradation. For the temperature dependence studies, the sample temperature was controlled by flowing either cooled (∼10 °C) or heated (∼40 °C) N2 gas over the Raman cell. Samples were equilibrated at the respective temperatures for 30 - 60 min prior to data acquisition. Typical data acquisition times ranged from 30 to 60 min, except for the L1 H-NOX NO-complex, which was signal-averaged for 3 h because of a high fluorescence background. Electronic absorption spectra were obtained both before and after the Raman experiments to ensure that sample integrity was maintained. Raman spectra were corrected for wavelength dependence of the spectrometer efficiency, and cyclohexane was used for instrument calibration. The reported frequencies are accurate to ±2 cm−1, and the resolution of the spectra is 8 cm−1. For each Raman spectrum, the raw data were base-line-corrected, and the buffer background signal was subtracted.Extinction Coefficient Determination—The extinction coefficients were determined similarly to the methods described previously for Tt H-NOX and Vc H-NOX (2Karow D.S. Pan D. Tran R. Pellicena P. Presley A. Mathies R.A. Marletta M.A. Biochemistry. 2004; 43: 10203-10211Crossref PubMed Scopus (161) Google Scholar). Specifically, for the 6-coordinate FeII-NO complex of L2 H-NOX, the electronic spectra of various dilutions of a sample of the aerobic FeII-NO complex (prepared as described above) at 0 °C were recorded. The electronic spectra of dilutions of a sample of horse heart metmyoglobin (ϵ409 nm = 181 cm−1 mm−1) were also recorded and used as a standard for heme concentration. The heme content of each sample was determined by HPLC (Hewlett Packard Series II 1090 HPLC with a diode array detector). Each sample (75 μl) was applied to a C4 column (250 × 4.6 mm, 5 μm; Vydac) that had been equilibrated with 0.1% trifluoroacetic acid. The column was developed with a linear gradient of 0-75% acetonitrile over 25 min followed by a linear gradient of 75-100% acetonitrile over 5 min. The column was washed and re-equilibrated between runs with a gradient of 100-0% acetonitrile over 3 min followed by 5 min of 100% aqueous phase (0.1% trifluoroacetic acid). The flow rate was 1 ml/min.NO Dissociation Rate—NO dissociation rates were measured as described previously (5Boon E.M. Huang S.H. Marletta M.A. Nat. Chem. Biol. 2005; 1: 53-59Crossref PubMed Scopus (167) Google Scholar). Briefly, FeII-NO complexes of protein (5 μm heme final concentration) diluted in anaerobic 50 mm triethanolamine, 50 mm NaCl, pH 7.5, buffer were rapidly mixed with a saturated carbon monoxide and 30 mm (final concentration) dithionite trap (Na2S2O4) in the same buffer (anaerobic) (11Kharitonov V.G. Sharma V.S. Magde D. Koesling D. Biochemistry. 1997; 36: 6814-6818Crossref PubMed Scopus (163) Google Scholar, 12Moore E.G. Gibson Q.H. J. Biol. Chem. 1976; 251: 2788-2794Abstract Full Text PDF PubMed Google Scholar). It has been established previously that CO binding is not rate-limiting in these experiments (11Kharitonov V.G. Sharma V.S. Magde D. Koesling D. Biochemistry. 1997; 36: 6814-6818Crossref PubMed Scopus (163) Google Scholar); this was confirmed in experiments using only 30 mm Na2S2O4 without CO as a trap. Data were acquired by scanning periodically on a Cary 3E spectrophotometer equipped with a Neslab RTE-100 constant temperature bath set to varying temperatures (0-70 °C). The dissociation of NO from the heme was monitored as the formation of the FeII-CO complex at 423 nm. Difference spectra were calculated by subtracting the first scan from each subsequent scan. The NO dissociation rate was determined from the increase in absorbance at 423 nm versus time and fit with a single or two parallel exponentials of the form f(x) = A × (1 - e−kx). Each experiment was performed a minimum of six times, and the resulting rates were averaged. The dissociation rates measured are independent of CO and dithionite concentration (3, 30, and 300 mm dithionite were tested).RESULTSElectronic Spectroscopy—UV-visible spectra of L2 H-NOX, L1 H-NOX, and Np H-NOX proteins as the FeII-unligated, FeII-CO, and FeII-NO complexes at room temperature are shown in Fig. 1 and compared with sGC, Tt H-NOX, and other histidyl-ligated heme proteins in Table 1. Interestingly, although the FeII-unligated and CO complexes of each of these proteins are similar to sGC and all other H-NOX proteins characterized to date (Table 1), there are some significant differences in the FeII-NO complexes. Specifically, L1 H-NOX forms a 5-coordinate NO complex with a characteristic Soret absorbance maximum at 398 nm (Fig. 1B) like sGC, but L2 H-NOX (Fig. 1C) and Np H-NOX (Fig. 1A) appear to be composed of a mixture of 5- and 6-coordinate FeII-NO complexes at 20 °C (399 and 416 nm, respectively). L2 H-NOX is a relatively evenly distributed mixture, whereas Np H-NOX appears to be primarily a 6-coordinate complex with a small shoulder corresponding to the 5-coordinate complex. Given the degree of homology of these proteins to sGC (Np H-NOX is 39% identical), which exclusively forms a 5-coordinate FeII-NO complex, an additional investigation into the NO-binding characteristics of these proteins was carried out.TABLE 1UV-visible peak positions (nm (at 20 °C)) for various FeII protein complexesProteinSoretβαRef.FeII unligated complexsGC43155536Stone J.R. Marletta M.A. Biochemistry. 1994; 33: 5636-5640Crossref PubMed Scopus (601) Google ScholarTt H-NOX4315652Karow D.S. Pan D. Tran R. Pellicena P. Presley A. Mathies R.A. Marletta M.A. Biochemistry. 2004; 43: 10203-10211Crossref PubMed Scopus (161) Google ScholarVc H-NOX4295682Karow D.S. Pan D. Tran R. Pellicena P. Presley A. Mathies R.A. Marletta M.A. Biochemistry. 2004; 43: 10203-10211Crossref PubMed Scopus (161) Google ScholarNp H-NOX430555This workL1 H-NOX428557This workL2 H-NOX428557This workL2 F142Y428557This workHb43055537Di Iorio E.E. Hemoglobins. Academic Press, New York1981Google ScholarFeII-CO complexsGC42354156736Stone J.R. Marletta M.A. Biochemistry. 1994; 33: 5636-5640Crossref PubMed Scopus (601) Google ScholarTt H-NOX4245445652Karow D.S. Pan D. Tran R. Pellicena P. Presley A. Mathies R.A. Marletta M.A. Biochemistry. 2004; 43: 10203-10211Crossref PubMed Scopus (161) Google ScholarVc H-NOX4235415662Karow D.S. Pan D. Tran R. Pellicena P. Presley A. Mathies R.A. Marletta M.A. Biochemistry. 2004; 43: 10203-10211Crossref PubMed Scopus (161) Google ScholarNp H-NOX423539566This workL1 H-NOX420540566This workL2 H-NOX423540571This workL2 F142Y422539569This workHb41954056937Di Iorio E.E. Hemoglobins. Academic Press, New York1981Google ScholarFeII-NO complexsGC39853757236Stone J.R. Marletta M.A. Biochemistry. 1994; 33: 5636-5640Crossref PubMed Scopus (601) Google ScholarTt H-NOX4205475752Karow D.S. Pan D. Tran R. Pellicena P. Presley A. Mathies R.A. Marletta M.A. Biochemistry. 2004; 43: 10203-10211Crossref PubMed Scopus (161) Google ScholarVc H-NOX3985405732Karow D.S. Pan D. Tran R. Pellicena P. Presley A. Mathies R.A. Marletta M.A. Biochemistry. 2004; 43: 10203-10211Crossref PubMed Scopus (161) Google ScholarNp H-NOX416/400543576This workL1 H-NOX398540571This workL2 H-NOX399/416544575This workL2 F142Y417544578This workHb41854557537Di Iorio E.E. Hemoglobins. Academic Press, New York1981Google Scholar Open table in a new tab Resonance Raman Spectroscopy—The resonance Raman spectra of the FeII-NO complexes of L2 H-NOX, L1 H-NOX, and Np H-NOX are shown in Fig. 2. Table 2 details the assignment of the major heme skeletal modes and compares them to sGC, Tt H-NOX, and other histidyl-ligated heme proteins. The Raman spectra confirm observations made in the electronic absorption spectra that L1 H-NOX forms a 5-coordinate NO complex, whereas L2 H-NOX and Np H-NOX have mixed coordination states, the specifics of which are discussed below. The π-electron density marker, ν4, is used to determine the oxidation state of the heme. The spin and coordination state markers, ν3, ν2, and ν10, are sensitive to the core size of the heme macrocycle (13Spiro T.G. Li X.-Y. Biological Applications of Raman Spectroscopy. John Wiley & Sons, Inc., New York1988Google Scholar). Typical frequency shifts of 3-15 cm−1 are often observed upon switching from 5- to 6-coordinate complexes for some of these skeletal markers.FIGURE 2Resonance Raman spectra of H-NOX domains as the FeII-NO complex at 20 °C. Low frequency resonance Raman spectrum of L2 H-NOX (20 μm) (A), L1 H-NOX (25 μm) (B), and Np H-NOX (40 μm) (C). High frequency resonance Raman spectrum of L2 H-NOX (20 μm) (D), L1 H-NOX (25 μm) (E), and Np H-NOX (40 μm) (F). Heme concentration is indicated in parentheses.View Large Image Figure ViewerDownload Hi-res image Download (PPT)TABLE 2Heme skeletal vibrations and vibrational modes (cm−1) for some FeII-NO proteinsProteinCNν10ν2ν3ν4νFe-NRef.sGC5164615841509137552514Tomita T. Ogura T. Tsuyama S. Imai Y. Kitagawa T. Biochemistry. 1997; 36: 10155-10160Crossref PubMed Scopus (89) Google Scholar, 31Deinum G. Stone J.R. Babcock G.T. Marletta M.A. Biochemistry. 1996; 35: 1540-1547Crossref PubMed Scopus (183) Google ScholarTt H-NOX616251580149613705532Karow D.S. Pan D. Tran R. Pellicena P. Presley A. Mathies R.A. Marletta M.A. Biochemistry. 2004; 43: 10203-10211Crossref PubMed Scopus (161) Google ScholarVc H-NOX516401580150513725232Karow D.S. Pan D. Tran R. Pellicena P. Presley A. Mathies R.A. Marletta M.A. Biochemistry. 2004; 43: 10203-10211Crossref PubMed Scopus (161) Google ScholarNp H-NOX51645158015061372528This workNp H-NOX61632158014991372559This workL1 H-NOX51643158115071373522This workL2 H-NOX51646158315081375521This workL2 H-NOX61633158315021375550This workMyoglobin6163815841501137555415Tsubaki M. Yu N.T. Biochemistry. 1982; 21: 1140-1144Crossref PubMed Scopus (67) Google ScholarAx cyt c′aAx cyt c′ indicates cytochrome c′ from Alcaligenes xylosoxidans; Rm FixLN indicates heme domain of FixL from Rhizogium meliloti5164115921506137352617Andrew C.R. Green E.L. Lawson D.M. Eady R.R. Biochemistry. 2001; 40: 4115-4122Crossref PubMed Scopus (77) Google ScholarAx cyt c′616381596150457916Andrew C.R. George S.J. Lawson D.M. Eady R.R. Biochemistry. 2002; 41: 2353-2360Crossref PubMed Scopus (77) Google ScholarRm FixLNaAx cyt c′ indicates cytochrome c′ from Alcaligenes xylosoxidans; Rm FixLN indicates heme domain of FixL from Rhizogium meliloti51646150952518Lukat-Rodgers G.S. Rodgers K.R. Biochemistry. 1997; 36: 4178-4187Crossref PubMed Scopus (78) Google ScholarRm FixLN61632149855818Lukat-Rodgers G.S. Rodgers K.R. Biochemistry. 1997; 36: 4178-4187Crossref PubMed Scopus (78) Google Scholara Ax cyt c′ indicates cytochrome c′ from Alcaligenes xylosoxidans; Rm FixLN indicates heme domain of FixL from Rhizogium meliloti Open table in a new tab In the low frequency spectrum of L2 H-NOX (Fig. 2A), two bands at 521 and 550 cm−1 are assigned to the Fe-N stretching modes (νFe-N) for 5- and 6-coordinate NO complexes, respectively, based on their similarity to values reported previously for this vibrational mode (2Karow D.S. Pan D. Tran R. Pellicena P. Presley A. Mathies R.A. Marletta M.A. Biochemistry. 2004; 43: 10203-10211Crossref PubMed Scopus (161) Google Scholar, 14Tomita T. Ogura T. Tsuyama S. Imai Y. Kitagawa T. Biochemistry. 1997; 36: 10155-10160Crossref PubMed Scopus (89) Google Scholar, 15Tsubaki M. Yu N.T. Biochemistry. 1982; 21: 1140-1144Crossref PubMed Scopus (67) Google Scholar). In the high frequency region of L2 H-NOX (Fig. 2D), ν3, ν2, and ν10 are primarily positioned at 1502, 1583, and 1633 cm−1, respectively, and are characteristic of histidyl-ligated, 6-coordinate, low spin, FeII-NO heme complexes such as myoglobin and Tt H-NOX (Table 2). However, there are also shoulders that appear at 1508 and 1646 cm−1, which correspond well to the marker values typically observed for the 5-coordinate nitrosyl heme complexes, such as sGC (Table 2). Together, these spectra strongly suggest that L2 H-NOX is a mixture of 5- and 6-coordinate complexes under room temperature conditions (∼20 °C).Although the signal-to-noise ratio is lower for L1 H-NOX because of a high fluorescence background (Fig. 2, B and E), the main skeletal modes are still discernible. Fig. 2B shows a band at 522 cm−1 assigned to the Fe-N stretching mode based on similarity to the 5-coordinate ferrous nitrosyl complexes in sGC (525 cm−1) and V. cholerae H-NOX (Vc H-NOX; 523 cm−1). The high frequency region of L1 H-NOX shows ν3, ν2, and ν10 at 1507, 1581, and 1643 cm−1, respectively (Fig. 2E). These observed vibrational frequencies for L1 H-NOX are consistent with other 5-coordinate FeII-NO heme complexes (2Karow D.S. Pan D. Tran R. Pellicena P. Presley A. Mathies R.A. Marletta M.A. Biochemistry. 2004; 43: 10203-10211Crossref PubMed Scopus (161) Google Scholar, 14Tomita T. Ogura T. Tsuyama S. Imai Y. Kitagawa T. Biochemistry. 1997; 36: 10155-10160Crossref PubMed Scopus (89) Google Scholar, 15Tsubaki M. Yu N.T. Biochemistry. 1982; 21: 1140-1144Crossref PubMed Scopus (67) Google Scholar) and are clearly shifted from the observed skeletal markers for 6-coordinate nitrosyl complexes (Table 2).Fig. 2, C and F, presents the low and high frequency resonance Raman spectra for the FeII-NO complex of Np H-NOX. Our results indicate that the nitrosyl complex for Np H-NOX is primarily 6-coordinate, with νFe-N assigned at 559 cm−1 based on previous work (6Karow D.S. Davis J.H. Marletta M.A. Biochemistry. 2005; 44: 16266-16274Crossref PubMed Scopus (67) Google Scholar, 15Tsubaki M. Yu N.T. Biochemistry. 1982; 21: 1140-1144Crossref PubMed Scopus (67) Google Scholar) (isotope data not shown); the spin and coordination state markers were observed at 1499, 1580, and 1632 cm−1 for ν3, ν2, and ν10, respectively. Closer inspection of the high frequency spectra show small, broad shoulder bands for both ν3 and ν10 at 1506 and 1645 cm−1, supporting the appearance of a mixture of 5- and 6-coordinate complexes in the electronic absorption spectra at ∼20 °C.Temperature Dependence of the 5- and 6-Coordinate NO Complexes—To investigate the mixture of 5- and 6-coordinate complexes, temperature-dependent studies of the FeII-NO complex of Np H-NOX and L2 H-NOX were carried out. Electronic absorption spectra indicate that both Np H-NOX and L2 H-NOX are in equilibrium between 5- and 6-coordinate complexes at physiologically relevant temperatures, as indicated by the Soret λmax shift with temperature (Fig. 3). The temperature was varied between 1 and 45 °C in the same sample several times showing that this 5- to 6-coordination behavior was fully reversible. Furthermore, the temperature-dependent coordination states are independent of NO concentration for both H-NOX proteins; no excess NO was added to any of the samples; the only NO in solution was that bound to the heme at the beginning of the experiment. The same results are obtained when the experiment is carried out anaerobically, indicating that O2 has no effect on this process.FIGURE 3Temperature-dependent electronic absorption spectra of the FeII-NO complexes of H-NOX domains. Np H-NOX (12 μm) (A) and L2 H-NOX (7 μm) (B). Heme concentration is indicated in parentheses. Temperatures for Np H-NOX are 4 °C (—), 10 °C (···), 20 °C (- - -), and 37 °C (- - -). The corresponding temperatures for L2 H-NOX are those shown with the same dash and color patterns using thick lines. In addition, 1, 15, 25, 30, 35, 40, and 45 °C are illustrated using thin lines. The 6-coordinate complex at 416 nm decreases simultaneously with increasing 5-coordinate complex at 399 nm as the temperature increases from 1 to 45 °C. This temperature dependence implicates Fe-His bond formation as an exothermic reaction.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Quantification of the amount of 5- and 6-coordinate complexes present at each temperature requires measurement of the extinction coefficient; however, the only complex that can be isolated to accurately measure the extinction coefficient was L2 H-NOX at 0 °C. Thus the extinction coefficient of the 6-coordinate NO complex of L2 H-NOX was determined to be 139 mm−1 cm−1, which was then used to estimate the mixture of each complex at a given temperature. At 40 °C, there is approximately a 50% mixture of each coordination state in the FeII-NO complex of L2 H-NOX. Even at −12 °C, L1 H-NOX shows no evidence of a 6-coordinate NO complex, although it is expected that at a sufficiently low temperature, the NO complex of L1 H-NOX would also convert to the 6-coordinate species.The temperature-dependent behavior was further investigated for L2 H-NOX between ∼10 and ∼40 °C by using resonance Raman spectroscopy. The high frequency spectra obtained at ∼10 °C for L2 H-NOX show ν3, ν2, and ν10 at 1500, 1586, and 1633 cm−1, respectively. In corroboration with the electronic absorption spectra (Fig. 3B), these observed vibrational frequencies show a shift in the equilibrium population toward a 6-coordinate complex with decreasing temperature. The spin state marker, ν3, is clearly split with overlapped bands at 1502 and 1508 cm−1 upon increasing the temperature to ∼20 °C (Fig. 4, B and E). The vibrational frequency for ν3 increases to 1510 cm−1 when the temperature is raised to ∼40 °C, supporting an equilibrium shift toward the 5-coordinate complex (Fig. 4, C and F). A temperature-dependent shoulder is also observed for ν10 at 1646 cm−1 with increasing temperature (Fig. 4, D-F). This shift in the vibrational frequency for ν10 has similarly been observed for 5- and 6-coordinate heme-NO complexes in cytochrome c′ (16Andrew C.R. George S.J. Lawson D.M. Eady R.R. Biochemistry. 2002; 41: 2353-2360Crossref PubMed Scopus (77) Google Scholar, 17Andrew C.R. Green E.L. Lawson D.M. Eady R.R. Biochemistry. 2001; 40: 4115-4122Crossref PubMed Scopus (77) Google Scholar) and FixLN (18Lukat-Rodgers G.S. Rodgers K.R. Biochemistry. 1997; 36: 4178-4187Crossref PubMed Scopus (78) Google Scholar).FIGURE 4Temperature-dependent resonance Raman spectra of the FeII-NO complex in L2 H-NOX. High frequency resonance Raman spectrum at ∼10 °C (A), ∼20 °C (B), and
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