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• W2034407646 abstract "Regulation of soluble guanylate cyclase (sGC), the primary NO receptor, is linked to NO binding to the prosthetic heme group. Recent studies have demonstrated that the degree and duration of sGC activation depend on the presence and ratio of purine nucleotides and on the presence of excess NO. We measured NO dissociation from full-length α1β1 sGC, and the constructs β1(1–194), β1(1–385), and β2(1–217), at 37 and 10 °C with and without the substrate analogue guanosine-5′-[(α,β-methylene]triphosphate (GMPCPP) or the activator 3-(5′-hydroxymethyl-3′-furyl)-1-benzylindazole (YC-1). NO dissociation from each construct was complex, requiring two exponentials to fit the data. Decreasing the temperature decreased the contribution of the faster exponential for all constructs. Inclusion of YC-1 moderately accelerated NO dissociation from sGC and β2(1–217) at 37 °C and dramatically accelerated NO dissociation from sGC at 10 °C. The presence of GMPCPP also dramatically accelerated NO dissociation from sGC at 10 °C. This acceleration is due to increases in the observed rate for each exponential and in the contribution of the faster exponential. Increases in the contribution of the faster exponential correlated with higher activation of sGC by NO. These data indicate that the sGC ferrous-nitrosyl complex adopts two 5-coordinate conformations, a lower activity “closed” form, which releases NO slowly, and a higher activity “open” form, which releases NO rapidly. The ratio of these two species affects the overall rate of NO dissociation. These results have implications for the function of sGC in vivo, where there is evidence for two NO-regulated activity states. Regulation of soluble guanylate cyclase (sGC), the primary NO receptor, is linked to NO binding to the prosthetic heme group. Recent studies have demonstrated that the degree and duration of sGC activation depend on the presence and ratio of purine nucleotides and on the presence of excess NO. We measured NO dissociation from full-length α1β1 sGC, and the constructs β1(1–194), β1(1–385), and β2(1–217), at 37 and 10 °C with and without the substrate analogue guanosine-5′-[(α,β-methylene]triphosphate (GMPCPP) or the activator 3-(5′-hydroxymethyl-3′-furyl)-1-benzylindazole (YC-1). NO dissociation from each construct was complex, requiring two exponentials to fit the data. Decreasing the temperature decreased the contribution of the faster exponential for all constructs. Inclusion of YC-1 moderately accelerated NO dissociation from sGC and β2(1–217) at 37 °C and dramatically accelerated NO dissociation from sGC at 10 °C. The presence of GMPCPP also dramatically accelerated NO dissociation from sGC at 10 °C. This acceleration is due to increases in the observed rate for each exponential and in the contribution of the faster exponential. Increases in the contribution of the faster exponential correlated with higher activation of sGC by NO. These data indicate that the sGC ferrous-nitrosyl complex adopts two 5-coordinate conformations, a lower activity “closed” form, which releases NO slowly, and a higher activity “open” form, which releases NO rapidly. The ratio of these two species affects the overall rate of NO dissociation. These results have implications for the function of sGC in vivo, where there is evidence for two NO-regulated activity states. Soluble guanylate cyclase (sGC) 4The abbreviations used are: sGC, soluble guanylate cyclase; NO, nitric oxide; β1(1–194), the first 194 amino acids of the β1 subunit of sGC; β1(1–385), the first 385 amino acids of the β1 subunit of sGC; β2(1–217), the first 217 amino acids of the β2 subunit of sGC; H-NOX, heme-nitric oxide/oxygen binding domain; YC-1, 3-(5′-hydroxymethyl-3′-furyl)-1-benzylindazole; GMPCPP, guanosine-5′-[(α,β-methylene]triphosphate; DEA/NO, diethylammonium (Z)-1-(N,N-diethylamino)diazen-1-ium-1,2-diolate; DTT, dithiothreitol; Me2SO, dimethyl sulfoxide. is the best characterized physiological receptor for the gaseous signaling agent nitric oxide (NO) (1Ignarro L.J. Semin. Hematol. 1989; 26: 63-76PubMed Google Scholar, 2Yuen P.S. Garbers D.L. Annu. Rev. Neurosci. 1992; 15: 193-225Crossref PubMed Scopus (86) Google Scholar, 3Denninger J.W. Marletta M.A. Biochim. Biophys. Acta. 1999; 1411: 334-350Crossref PubMed Scopus (883) Google Scholar, 4Wedel B. Garbers D. Annu. Rev. Physiol. 2001; 63: 215-233Crossref PubMed Scopus (111) Google Scholar, 5Friebe A. Koesling D. Circ. Res. 2003; 93: 96-105Crossref PubMed Scopus (427) Google Scholar). In response to NO, sGC produces the second messenger cGMP, modulating physiological processes such as neurotransmission and vasodilation (6Munzel T. Feil R. Mulsch A. Lohmann S.M. Hofmann F. Walter U. Circulation. 2003; 108: 2172-2183Crossref PubMed Scopus (274) Google Scholar). The α1β1 sGC heterodimer is activated several hundredfold above the basal level by the binding of NO to the heme of the β1 H-NOX domain (7Stone J.R. Marletta M.A. Biochemistry. 1996; 35: 1093-1099Crossref PubMed Scopus (290) Google Scholar, 8Brandish P.E. Buechler W. Marletta M.A. Biochemistry. 1998; 37: 16898-16907Crossref PubMed Scopus (87) Google Scholar, 9Hoenicka M. Becker E.M. Apeler H. Sirichoke T. Schroder H. Gerzer R. Stasch J.P. J. Mol. Med. 1999; 77: 14-23Crossref PubMed Scopus (117) Google Scholar), a conserved domain of unique structure (10Iyer L.M. Anantharaman V. Aravind L. BMC Genomics. 2003; 4: 5Crossref PubMed Scopus (156) Google Scholar, 11Pellicena 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 (251) Google Scholar, 12Nioche P. Berka V. Vipond J. Minton N. Tsai A.L. Raman C.S. Science. 2004; 306: 1550-1553Crossref PubMed Scopus (177) Google Scholar). However, it remains unclear how this binding event is translated into increased catalytic activity. The mechanism by which sGC deactivation occurs has been a focus of much investigation (8Brandish P.E. Buechler W. Marletta M.A. Biochemistry. 1998; 37: 16898-16907Crossref PubMed Scopus (87) Google Scholar, 13Margulis A. Sitaramayya A. Biochemistry. 2000; 39: 1034-1039Crossref PubMed Scopus (45) Google Scholar, 14Russwurm M. Mergia E. Mullershausen F. Koesling D. J. Biol. Chem. 2002; 277: 24883-24888Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar). Initially thought to result from simple dissociation of NO from the heme, the deactivation process has turned out to be more complicated. In fact, regulation of sGC by NO has been shown to involve a complex interplay between binding of NO to the heme and to non-heme sites as well as allosteric regulation by GTP, also a substrate, and ATP, a reporter for the energy status of the cell (15Cary S.P.L. Winger J.A. Derbyshire E.R. Marletta M.A. Trends Biochem. Sci. 2006; 31: 231-239Abstract Full Text Full Text PDF PubMed Scopus (189) Google Scholar). In integrating the inputs from all these signals, sGC has been shown to have a remarkable attribute, the ability to exist in a stable low activity state or a transient high activity state, both containing NO bound to the heme (16Cary S.P.L. Winger J.A. Marletta M.A. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 13064-13069Crossref PubMed Scopus (126) Google Scholar, 17Russwurm M. Koesling D. EMBO J. 2004; 23: 4443-4450Crossref PubMed Scopus (169) Google Scholar). Many questions concerning the existence and characteristics of these two sGC heme-NO species, and how they might be regulated by NO and nucleotides, remain to be addressed. In addition to responding to cellular inputs, sGC can be activated by a class of small molecules exemplified by 3-(5′-hydroxymethyl-3′-furyl)-1-benzylindazole (YC-1). These molecules not only activate sGC in the absence of heme ligands but also synergize with the NO- and CO-bound forms of the enzyme to reach maximal activity. There has been much speculation about the binding site and mechanism of action of YC-1 (18Hering K.W. Artz J.D. Pearson W.H. Marletta M.A. Bioorg. Med. Chem. Lett. 2006; 16: 618-621Crossref PubMed Scopus (20) Google Scholar, 19Lamothe M. Chang F.J. Balashova N. Shirokov R. Beuve A. Biochemistry. 2004; 43: 3039-3048Crossref PubMed Scopus (82) Google Scholar, 20Stasch J.P. Becker E.M. Alonso-Alija C. Apeler H. Dembowsky K. Feurer A. Gerzer R. Minuth T. Perzborn E. Pleiss U. Schroder H. Schroeder W. Stahl E. Steinke W. Straub A. Schramm M. Nature. 2001; 410: 212-215Crossref PubMed Scopus (482) Google Scholar); however, experimental results remain inconclusive. By using purified sGC, and sGC heme domain constructs (Fig. 1) that possess heme characteristics similar to those of the full-length α1β1 enzyme (21Karow D.S. Pan D. Davis J.H. Behrends S. Mathies R.A. Marletta M.A. Biochemistry. 2005; 44: 16266-16274Crossref PubMed Scopus (67) Google Scholar, 22Zhao Y. Marletta M.A. Biochemistry. 1997; 36: 15959-15964Crossref PubMed Scopus (107) Google Scholar), we carried out spectroscopic and kinetic analyses of NO dissociation in the absence and presence of YC-1 and the substrate analogue guanosine-5′-[(α,β-methylene]triphosphate (GMPCPP), as well as activity studies with YC-1. Dissociation was found to exhibit two exponential phases, the relative contributions of which could be differentially affected by YC-1, GMPCPP, or changes in temperature. We propose a model for dissociation of NO from sGC involving the existence of two 5-coordinate sGC heme-NO species indistinguishable by electronic absorption spectroscopy but clearly different in their ability to release NO from the heme; this model is discussed in context of the current hypothesis for regulation of sGC by NO and nucleotides. Materials—Primers were obtained from Invitrogen. All restriction enzymes were from New England Biolabs. Sf9 cells were obtained from the Department of Molecular and Cell Biology Tissue Culture Facility, University of California, Berkeley. Rat lung sGC α1 and β1 cDNAs were provided by Dr. Masaki Nakane, Abbott. H-NOX domain constructs from rat sGC β1 and β2 subunits (β1(1–194), β1(1–385), and β2(1–217)) were purified as described (21Karow D.S. Pan D. Davis J.H. Behrends S. Mathies R.A. Marletta M.A. Biochemistry. 2005; 44: 16266-16274Crossref PubMed Scopus (67) Google Scholar, 23Zhao Y. Brandish P.E. DiValentin M. Schelvis J.P. Babcock G.T. Marletta M.A. Biochemistry. 2000; 39: 10848-10854Crossref PubMed Scopus (184) Google Scholar). 3-(5′-Hydroxymethyl-3′-furyl)-1-benzylindazole (YC-1) and the NO donor diethylammonium (Z)-1-(N,N-diethylamino)diazen-1-ium-1,2-diolate (DEA/NO) were purchased from Cayman Chemical Co. CO gas was from Praxair. GMPCPP was from Jena Biosciences. All other chemicals were purchased from Sigma unless otherwise stated. Baculovirus Construction—Rat sGC β1 cDNA was inserted between the NotI and XbaI sites of the plasmid pFastBac1 (Invitrogen) to generate pFastBac1/sGCβ1. PCR was used to insert an in-frame C-terminal RGS-H6 tag in front of the stop codon of the α1 cDNA. The forward primer was 5′-TGGCGGCCGCAAGGAGGAAACCAC-3′, and the reverse primer was 5′-CGTCTAGATTAGTGGTGGTGGTGGTGGTGAGATCCTCTATCTACCCCTGATGCTTTGCCTAAGAAGTTAGCGTTTCC-3′. PCR products were sequenced to confirm the presence of the desired changes (University of Michigan Biomedical Research Core Facilities). The H6sGC α1 gene was inserted between the NotI and XbaI sites of pFastBac1 to generate the construct pFastBac1/sGCα1. The Bac-to-Bac baculovirus expression system (Invitrogen) was used to generate recombinant baculoviruses from pFastBac1/sGCα1 and pFastBac1/sGCβ1 according to the manufacturer's protocol. High titer stocks of recombinant baculoviruses were prepared by standard methods. Optimization of the amount of each virus used for protein production was carried out as described previously (23Zhao Y. Brandish P.E. DiValentin M. Schelvis J.P. Babcock G.T. Marletta M.A. Biochemistry. 2000; 39: 10848-10854Crossref PubMed Scopus (184) Google Scholar). Cell Culture and Production of Recombinant sGC—Sf9 cells were cultured in Ex-Cell 420 insect serum-free medium (JRH Biosciences) supplemented with 10% fetal calf serum (Hyclone) and 1% antibiotic/antimycotic (Invitrogen) at 28 °C. Cultures were grown in 2800-ml Fernbach flasks with shaking at 135 rpm. Cells were subcultured between 0.7 × 106 and 5 × 106 cells/ml. Cell density and viability were determined by trypan blue exclusion using a hemocytometer. For protein expression, 1-liter cultures of Sf9 cells at a density of 1.5–2 × 106 cells/ml in 2800-ml Fernbach flasks were infected with H6α1 and β1 recombinant viruses. Cells were harvested 3 days post-infection by centrifugation, and the pellet was stored at –80 °C. Purification of Recombinant sGC—All manipulations were carried out at 4 °C. Frozen cell pellets from 5-liter expression cultures were thawed on ice and resuspended in buffer A (50 mm KH2PO4, pH 8.0, 200 mm NaCl, 5 mm β-mercaptoethanol, 1 mm imidazole, 1 mm Pefabloc (Pentapharm), 1 mm benzamidine, 5% glycerol) plus Complete EDTA-free protease inhibitor mixture (Roche Applied Science). Resuspended cells were broken with a Bead Beater (BioSpec Products) using 0.1-mm diameter glass beads, and the lysate was centrifuged at 200,000 × g for 2 h. The supernatant was applied to a 2.5-ml column of nickel-nitrilotriacetic acid-agarose (Qiagen) equilibrated with buffer A at a flow rate of 1 ml/min using a BioLogic LP (Bio-Rad). The column was washed with buffer A until the A280 was stable, and then an aliquot of buffer A (25 ml) was brought to 1.2 m NaCl and applied to the column. The column was washed with 50 ml of 12.5 mm imidazole in buffer A and eluted with 25 ml of 125 mm imidazole in buffer A, collecting 2-ml fractions during the elution. Fractions containing sGC (identified by yellow color) were pooled, concentrated to 1–1.5 ml in a Vivaspin-20 50K filter (Vivascience), and exchanged into buffer B (25 mm triethanolamine, pH 7.4, 25 mm NaCl, 5 mm dithiothreitol)) on a PD-10 column (Amersham Biosciences). The sample was diluted to ∼7 ml with buffer B and applied to a 2-ml prepacked POROS HQ2 anion-exchange column (Applied Biosystems) at 2 ml/min using a BioLogic Duo Flow (Bio-Rad). The column was washed with 5 ml of buffer B and developed with a 35-ml 120–285 mm gradient of NaCl in buffer B, collecting 1-ml fractions. Fractions containing purified sGC (exhibiting an A278/A431 < 1.1) were pooled, concentrated in a Vivaspin-6 50K filter, drop-frozen in liquid N2, and stored in liquid N2. Protein purity was assessed by SDS-PAGE using pre-cast 10% Tris-glycine gels (Invitrogen) and was routinely greater than 95%. Protein concentrations were determined using the Bradford microassay (Bio-Rad) or calculated from the A431 using an extinction coefficient of 148,000 m–1 cm–1 (8Brandish P.E. Buechler W. Marletta M.A. Biochemistry. 1998; 37: 16898-16907Crossref PubMed Scopus (87) Google Scholar). Dissociation of NO from the Heme of β1(1–194), β1(1–385), β2(1–217), and sGC—The dissociation of NO from the heme of each H-NOX domain construct and sGC was measured at 37 and 10 °C using the CO/dithionite trapping method described previously (16Cary S.P.L. Winger J.A. Marletta M.A. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 13064-13069Crossref PubMed Scopus (126) Google Scholar, 24Kharitonov V.G. Sharma V.S. Magde D. Koesling D. Biochemistry. 1997; 36: 6814-6818Crossref PubMed Scopus (168) Google Scholar). The trapping solution was prepared as follows: a solution of sodium dithionite (Na2S2O4) in 50 mm HEPES, pH 7.4, 50 mm NaCl was prepared in a Teflon-sealed Reacti-Vial (Pierce) using an anaerobic chamber (Coy Laboratory Products). The solution was removed from the anaerobic chamber and saturated with CO by bubbling the gas through the solution for 10 min. Protein-NO complexes were formed by incubation with excess DEA/NO (in 10 mm NaOH) at 25 °C in 50 mm HEPES, pH 7.4, 50 mm NaCl for 10 min. Complete conversion to the nitrosyl species was verified by following the shift in the Soret maximum from 431 to 399 nm. Stock solutions of YC-1 were made in Me2SO. When present, YC-1 concentrations ranged from 0.96 to 96 μm, and the final concentration of Me2SO was 1%. Experiments with GMPCPP contained 10–1000 μm nucleotide (added before DEA/NO addition) and included 5 mm MgCl2, which alone had no effect on NO dissociation. Proteins were placed in a septum-sealed anaerobic cuvette and deoxygenated using an oxygen-scavenged gas train. A small amount of DEA/NO (∼3 eq) was added just before deoxygenation to maintain the nitrosyl species (any remainder was subsequently destroyed by the large excess of Na2S2O4 in the trapping solution). The head space of the anaerobic cuvette was replaced with CO, and the cuvette and trap solutions were equilibrated at assay temperature for 1 min. The reaction was initiated by addition of CO/dithionite solution to the anaerobic cuvette with a Hamilton gas-tight syringe and mixing. The final concentration of Na2S2O4 in the reaction mixture was 30 mm. Final protein concentrations were 1.9–2.5 μm for β1(1–194), β1(1–385), and β2(1–217), and 0.88–2.5 μm for sGC. Data collection was initiated ∼10 s after trap addition. The reaction was monitored by electronic absorption spectroscopy using a Cary 3E spectrophotometer equipped with a Neslab RTE-100 temperature controller. Data were collected over the range of 380–450 nm at 909 nm/min with a 1.5-nm data point interval. Spectra were recorded every 18 s for 5 min, every 1 min for 10 min, and every 2 min thereafter for a total of 3 h, or until the reaction was complete. A buffer base line was subtracted from each spectrum, and spectra were corrected for base-line drift by normalization to an isosbestic point at ∼410 nm. For data obtained in the absence of YC-1 or GMPCPP, difference spectra were obtained by subtraction of the time 0 spectrum from all subsequent spectra. To obtain difference spectra for data acquired in the presence of YC-1 or GMPCPP, a time 0 spectrum from a reaction carried out in the absence of either compound and containing an identical amount of protein was subtracted from all subsequent spectra, and all time points were offset by an amount corresponding to the mixing time for the experiment. Values for the change in absorbance at 423 nm (ΔA423; β1(1– 194) and β1(1–385)) or 424 nm (ΔA424; sGC and β2(1–217)) were extracted from the difference spectra and plotted versus time to obtain dissociation time courses for each experiment. Dissociation time courses were obtained in duplicate or triplicate, and each experiment was repeated 2–5 times over several days. Generally, because of the relative difficulty in obtaining large amounts of purified sGC, ΔA424 values for full-length sGC, which are proportional to the experimental protein concentrations, were smaller than for the heme domain constructs. YC-1 Activation of the sGC-NO Complex—End point assays were performed in triplicate at 10 and 37 °C as described previously (25Derbyshire E.R. Tran R. Mathies R.A. Marletta M.A. Biochemistry. 2005; 44: 16257-16265Crossref PubMed Scopus (17) Google Scholar). Stock solutions of DEA/NO (10 mm) were prepared in NaOH (10 mm). Stock solutions of YC-1 (15 mm) were prepared in Me2SO. Assay mixtures contained 0.2 μg of sGC in 50 mm HEPES, pH 7.4, 2 mm dithiothreitol, and 150 μm YC-1 where indicated. sGC was incubated with DEA/NO (100 μm) for 10 min at 25 °C and equilibrated at assay temperature for 1 min. Assays were initiated by addition of GTP and MgCl2 to 1 and 3 mm, respectively. Final assay volumes were 100 μl and contained 2% Me2SO, which did not affect enzyme activity. Reactions were quenched after 3 min by the addition of 400 μl of 125 mm Zn(CH3CO2)2 and 500 μl of 125 mm Na2CO3. cGMP quantification was carried out using a cGMP enzyme immunoassay kit, format B (Biomol), per the manufacturer's instructions. Each experiment was repeated four times to ensure reproducibility. Data Analysis and Statistics—Curve fitting, data analysis, and figure generation were carried out using Kaleidagraph (Synergy Software). The data from each dissociation experiment were fit to single or double exponentials as shown in Equations 2 and 3 under “Results” to obtain observed rate constants. To determine whether a single exponential or two exponentials best fit the data, the residuals from each fit were compared. Additionally, for each set of dissociation data, the fit to a single exponential was compared with the fit to two exponentials using the F test. A two-exponential fit was considered better than a one-exponential fit if p < 0.0001. For dissociation experiments, rates are expressed as means ± S.D. For activity assays, significant differences between the means were determined using the two-tailed t test; the significance level used was 0.05. Kinetic Considerations; Developing a Model for NO Dissociation from sGC—The binding of NO to the sGC heme has been shown to proceed through initial formation of a 6-coordinate intermediate, followed by rupture of the iron-histidine bond to form the final 5-coordinate ferrous nitrosyl complex (7Stone J.R. Marletta M.A. Biochemistry. 1996; 35: 1093-1099Crossref PubMed Scopus (290) Google Scholar, 26Zhao Y. Brandish P.E. Ballou D.P. Marletta M.A. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 14753-14758Crossref PubMed Scopus (301) Google Scholar, 27Makino R. Matsuda H. Obayashi E. Shiro Y. Iizuka T. Hori H. J. Biol. Chem. 1999; 274: 7714-7723Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar). The simplest mechanism for dissociation of NO from the 5-coordinate sGC heme-NO complex would be the reverse of NO binding as follows: rebinding of the proximal histidine ligand to form a 6-coordinate heme-NO intermediate followed by dissociation of NO to form a 5-coordinate histidyl-ligated heme, as shown in Scheme 1. heme-NO5C⇌kHdkHrheme-NO6C⇌kon[NO]koffheme5C+NOSCHEME 1 The values of kHr and kHd are the rate constants for rebinding and dissociation, respectively, of the proximal histidine ligand; kon is the rate constant for binding of NO to the heme, and koff is the rate constant for the dissociation of NO from the 6-coordinate heme-NO complex. In this study, we employed an NO trap, consisting of a sodium dithionite (Na2S2O4) solution saturated with CO (COsat), to obtain rates for dissociation uncomplicated by NO rebinding. This system has been used previously to determine the NO dissociation rates for a number of heme proteins (16Cary S.P.L. Winger J.A. Marletta M.A. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 13064-13069Crossref PubMed Scopus (126) Google Scholar, 24Kharitonov V.G. Sharma V.S. Magde D. Koesling D. Biochemistry. 1997; 36: 6814-6818Crossref PubMed Scopus (168) Google Scholar, 28Moore E.G. Gibson Q.H. J. Biol. Chem. 1976; 251: 2788-2794Abstract Full Text PDF PubMed Google Scholar) and functions by destroying dissociated NO through reaction with dithionite and by preventing NO rebinding by blocking the open heme coordination site with CO. At the concentrations of dithionite and CO used in these experiments, the reaction of NO with dithionite and the binding of CO to the vacated heme coordination site are not rate-limiting (Refs. 24Kharitonov V.G. Sharma V.S. Magde D. Koesling D. Biochemistry. 1997; 36: 6814-6818Crossref PubMed Scopus (168) Google Scholar and 29Boon E.M. Davis J.H. Tran R. Karow D.S. Huang S.H. Pan D. Miazgowicz M.M. Mathies R.A. Marletta M.A. J. Biol. Chem. 2006; 281: 21892-21902Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar and data not shown). Thus, under our NO-trapping conditions, the above reaction scheme simplifies to Scheme 2, heme-NO5C⇌kHdkHrheme-NO6C→koffheme-COSCHEME 2 where koff is the rate constant for the dissociation of NO from the 6-coordinate heme-NO complex to form the CO complex, which is irreversible because of the COsat/dithionite trap. Assuming a steady-state equilibrium between heme-NO5C and heme-NO6C, Equation 1 can be derived for the observed reaction rate after mixing (derivation of the first-order rate constant kobs for mechanisms similar to that described in Scheme 2 has been discussed in detail (29Boon E.M. Davis J.H. Tran R. Karow D.S. Huang S.H. Pan D. Miazgowicz M.M. Mathies R.A. Marletta M.A. J. Biol. Chem. 2006; 281: 21892-21902Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar, 30Trent J.T. Hvitved A.N. Hargrove M.S. Biochemistry. 2001; 40: 6155-6163Crossref PubMed Scopus (87) Google Scholar, 31Lowry T.M. John W.T. J. Chem. Soc. Trans. 1910; 97: 2634-2645Crossref Scopus (46) Google Scholar, 32Hvidt A. Nielsen S.O. Adv. Protein Chem. 1966; 21: 287-386Crossref PubMed Scopus (1024) Google Scholar, 33Smagghe B.J. Sarath G. Ross E. Hilbert J.L. Hargrove M.S. Biochemistry. 2006; 45: 561-570Crossref PubMed Scopus (67) Google Scholar)), kobs=kHrkoffkHr+kHd+koff(Eq. 1) A single exponential increase in the concentration of heme-CO is expected when starting from a uniform population of either heme-NO5C or heme-NO6C, as described by Equation 2, ΔAt=ΔAT(1-e-k1t)(Eq. 2) where ΔAt is the change in signal amplitude at time t; ΔAT is the total change in signal amplitude, and k1 is the observed reaction rate constant. Importantly, when starting from a mixture of heme-NO5C and heme-NO6C, if koff is faster than kHr, a two-exponential increase as described by Equation 3 is predicted, ΔAt=ΔA1(1-e-k1t)+ΔA2(1-e-k2t)(Eq. 3) where ΔAt is the change in signal amplitude at time t; ΔA1 and ΔA2 are the contributions of each exponential process to the total change in signal amplitude, and k1 and k2 are the observed rate constants for each process. That is exactly what is observed for dissociation of NO from several prokaryotic H-NOX domains, for which the heme-NO complexes have been demonstrated to exist as an equilibrium mixture of 5- and 6-coordinate states (29Boon E.M. Davis J.H. Tran R. Karow D.S. Huang S.H. Pan D. Miazgowicz M.M. Mathies R.A. Marletta M.A. J. Biol. Chem. 2006; 281: 21892-21902Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar). However, previous studies using electronic absorption and resonance Raman spectroscopy have demonstrated that for sGC and the sGC H-NOX constructs studied in this work, the heme-NO complex is exclusively 5-coordinate (21Karow D.S. Pan D. Davis J.H. Behrends S. Mathies R.A. Marletta M.A. Biochemistry. 2005; 44: 16266-16274Crossref PubMed Scopus (67) Google Scholar, 34Deinum G. Stone J.R. Babcock G.T. Marletta M.A. Biochemistry. 1996; 35: 1540-1547Crossref PubMed Scopus (186) Google Scholar, 35Stone J.R. Marletta M.A. Biochemistry. 1994; 33: 5636-5640Crossref PubMed Scopus (613) Google Scholar). From these observations, it can be inferred that there is no appreciable amount of heme-NO6C in a solution of NO-bound sGC, relegating heme-NO6C to the status of a transient intermediate in the dissociation reaction pathway. Thus, in order to accommodate any observed two-exponential dissociation of NO, Scheme 2 must be expanded to include an additional 5-coordinate heme-NO species, as shown in Scheme 3, heme-NO5C*⇌kCkOheme-NO5C⇌kHdkHrheme-NO6C→koffheme-COSCHEME 3 where heme-NO*5C and heme-NO5C are two 5-coordinate heme-NO species that are spectroscopically identical but kinetically distinct. In this mechanism, the 5-coordinate heme-NO complex of sGC exists as an equilibrium mixture of 5-coordinate heme-NO species (heme-NO*5C and heme-NO5C), with slow interconversion between the two forms compared with NO dissociation. A two-exponential NO dissociation time course would be observed if, upon mixing with the NO trap, NO dissociated from the heme-NO5C fraction with a kobs according to the reaction in Scheme 2, with the remainder of the dissociation reaction proceeding from the heme-NO*5C fraction, as indicated in Scheme 3, and dependent on the rate of interconversion between 5-coordinate species (kO – kC). Thus, the observation of two exponentials versus one in the dissociation of NO from a sGC H-NOX domain would indicate that NO dissociates from a mixture of 5-coordinate heme-NO species, with ΔA1 and ΔA2 proportional to the amount of each species at the start of the dissociation reaction. Dissociation of NO from sGC and sGC H-NOX Domain Constructs at 37 °C—In the presence of the NO trap (COsat/dithionite), dissociation of NO from the heme of sGC and sGC H-NOX domain constructs resulted in an increase in absorbance at 423–424 nm because of formation of the heme-CO complex. A representative set of difference spectra for dissociation at 37 °C from sGC, β1(1–194), β1(1–385), and β2(1–217) is shown in Fig. 2. The corresponding plots of ΔA423 or ΔA424 against time, shown in the top panels of Fig. 3, A–D, yield a dissociation time course for each construct. For each dissociation time course, the fits to both single and double exponentials (Equations 2 and 3) are shown. The residuals for each fit are plotted above each time course. In each case, examination of the residuals suggested that a two-exponential fit provided a better model for the data than a single exponential fit. A comparison of the one- and two-exponential fits using the F test supported the two-exponential fit as the better model in each case (p < 0.0001). The two rate constants obtained for each construct (a faster constant k1 and a slower constant k2, averaged from 2–4 experiments per construct) and the amplitudes of each corresponding phase (as a percent of the calculated total) are shown in Table 1. The observed data are consistent with a model where dissociation proceeds from an initial equilibrium mixture of two 5-coordinate heme-NO complexes, as outlined in Scheme 3. Accordingly, we propose that k1 corresponds to dissociation of NO from the heme-NO5C conformation at a rate equal to kobs in Equation 1, whereas k2 represents the observed rate of reaction, corresponding to kO – kC, that is limited by the slower conversion from heme-NO*5C to heme-NO5C.FIGURE 3Time courses for dissociation of NO from sGC and H-NOX domain constructs at 37 °C. Data were extracted from d" @default.
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• W2034407646 title "Dissociation of Nitric Oxide from Soluble Guanylate Cyclase and Heme-Nitric Oxide/Oxygen Binding Domain Constructs" @default.
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