The Role of Tyrosine 343 in Substrate Binding and Catalysis by Human Sulfite Oxidase
2004; Elsevier BV; Volume: 279; Issue: 15 Linguagem: Inglês
10.1074/jbc.m314288200
ISSN1083-351X
AutoresHeather L. Wilson, K.V. Rajagopalan,
Tópico(s)Ion Transport and Channel Regulation
ResumoIn the crystal structure of chicken sulfite oxidase, the residue Tyr322 (Tyr343 in human sulfite oxidase) was found to directly interact with a bound sulfate molecule and was proposed to have an important role in mediating the substrate specificity and catalytic activity of this molybdoprotein. In order to understand the role of this residue in the catalytic mechanism of sulfite oxidase, steady-state and stopped-flow analyses were performed on wild-type and Y343F human sulfite oxidase over the pH range 6–10. In steady-state assays of Y343F sulfite oxidase using cytochrome c as the electron acceptor, kcat was somewhat impaired (∼34% wild-type activity at pH 8.5), whereas the Kmsulfite showed a 5-fold increase over wild type. In rapid kinetic assays of the reductive half-reaction of wild-type human sulfite oxidase, kredheme changed very little over the entire pH range, with a significant increase in Kdsulfite at high pH. The kredheme of the Y343F variant was significantly impaired across the entire pH range, and unlike the wild-type protein, both kredheme and Kdsulfite were dependent on pH, with a significant increase in both kinetic parameters at high pH. Additionally, reduction of the molybdenum center by sulfite was directly measured for the first time in rapid reaction assays using sulfite oxidase lacking the N-terminal heme-containing domain. Reduction of the molybdenum center was quite fast (kredMo = 972 s-1 at pH 8.65 for wild-type protein), indicating that this is not the rate-limiting step in the catalytic cycle. Reduction of the molybdenum center of the Y343F variant by sulfite was more significantly impaired at high pH than at low pH. These results demonstrate that the Tyr343 residue is important for both substrate binding and oxidation of sulfite by sulfite oxidase. In the crystal structure of chicken sulfite oxidase, the residue Tyr322 (Tyr343 in human sulfite oxidase) was found to directly interact with a bound sulfate molecule and was proposed to have an important role in mediating the substrate specificity and catalytic activity of this molybdoprotein. In order to understand the role of this residue in the catalytic mechanism of sulfite oxidase, steady-state and stopped-flow analyses were performed on wild-type and Y343F human sulfite oxidase over the pH range 6–10. In steady-state assays of Y343F sulfite oxidase using cytochrome c as the electron acceptor, kcat was somewhat impaired (∼34% wild-type activity at pH 8.5), whereas the Kmsulfite showed a 5-fold increase over wild type. In rapid kinetic assays of the reductive half-reaction of wild-type human sulfite oxidase, kredheme changed very little over the entire pH range, with a significant increase in Kdsulfite at high pH. The kredheme of the Y343F variant was significantly impaired across the entire pH range, and unlike the wild-type protein, both kredheme and Kdsulfite were dependent on pH, with a significant increase in both kinetic parameters at high pH. Additionally, reduction of the molybdenum center by sulfite was directly measured for the first time in rapid reaction assays using sulfite oxidase lacking the N-terminal heme-containing domain. Reduction of the molybdenum center was quite fast (kredMo = 972 s-1 at pH 8.65 for wild-type protein), indicating that this is not the rate-limiting step in the catalytic cycle. Reduction of the molybdenum center of the Y343F variant by sulfite was more significantly impaired at high pH than at low pH. These results demonstrate that the Tyr343 residue is important for both substrate binding and oxidation of sulfite by sulfite oxidase. Mammalian sulfite oxidase (SO 1The abbreviations used are: SO, sulfite oxidase; cyt c, cytochrome c; MPT, molybdopterin; IET, intramolecular electron transfer; EXAFS, extended x-ray absorption fine structure; CEPT, coupled electron proton transfer; Bis-Tris, bis(2-hydroxyethyl) amino-tris(hydroxymethyl)methane; Bis-Tris propane, 1,3-bis[tris(hydroxymethyl)methylamino] propane. 1The abbreviations used are: SO, sulfite oxidase; cyt c, cytochrome c; MPT, molybdopterin; IET, intramolecular electron transfer; EXAFS, extended x-ray absorption fine structure; CEPT, coupled electron proton transfer; Bis-Tris, bis(2-hydroxyethyl) amino-tris(hydroxymethyl)methane; Bis-Tris propane, 1,3-bis[tris(hydroxymethyl)methylamino] propane.; EC 1.8.3.1) is an essential molybdoprotein that resides in the intermembrane space of the mitochondria and is responsible for the oxidation of sulfite to sulfate, the final reaction in the degradation of sulfur-containing compounds including the amino acids methionine and cysteine (1Rajagopalan K.V. Coughlan M.P. Molybdenum and Molybdenum-containing Enzymes. Pergamon Press, Oxford1980: 241-272Google Scholar, 2Rajagopalan K.V. Johnson J.L. Creighton T.E. Wiley Encyclopedia of Molecular Medicine. John Wiley and Sons, Inc., New York2002: 3048-3051Google Scholar, 3Enemark J.H. Cosper M.M. Sigel A. Sigel H. Metal Ions in Biological Systems. 39. Marcel Dekker, New York2002: 621-654Google Scholar, 4Schindelin H. Kisker C. Rajagopalan K.V. Adv. Protein Chem. 2001; 58: 47-94Crossref PubMed Scopus (66) Google Scholar). SO belongs to the family of proteins containing the molybdopterin (MPT) cofactor, which consists of a single molybdenum atom coordinated to a pterin derivative through a dithiolene group (4Schindelin H. Kisker C. Rajagopalan K.V. Adv. Protein Chem. 2001; 58: 47-94Crossref PubMed Scopus (66) Google Scholar, 5Wuebbens M.M. Rajagopalan K.V. J. Biol. Chem. 2003; 278: 14523-14532Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar). Deficiency of this enzyme due to either a defect in the MPT cofactor, or to a point mutation in the SO gene itself leads to severe neurological problems that often result in death at an early age (6Johnson J.L. Coyne K.E. Garrett R.M. Zabot M.T. Dorche C. Kisker C. Rajagopalan K.V. Hum. Mutat. 2002; 20: 74Crossref PubMed Scopus (57) Google Scholar, 7Johnson J.L. Rajagopalan K.V. Wadman S.K. Adv. Exp. Med. Biol. 1993; 338: 373-378Crossref PubMed Scopus (24) Google Scholar, 8Johnson J.L. Wuebbens M.M. Mandell R. Shih V.E. Biochem. Med. Metab. Biol. 1988; 40: 86-93Crossref PubMed Scopus (16) Google Scholar).Mammalian SO has been cloned, purified, and characterized from human and rat liver (9Garrett R.M. Rajagopalan K.V. J. Biol. Chem. 1994; 269: 272-276Abstract Full Text PDF PubMed Google Scholar, 10Garrett R.M. Bellissimo D.B. Rajagopalan K.V. Biochim. Biophys. Acta. 1995; 1262: 147-149Crossref PubMed Scopus (53) Google Scholar), and the crystal structure of native chicken liver SO has been solved at a resolution of 1.9 Å (11Kisker C. Schindelin H. Pacheco A. Wehbi W.A. Garrett R.M. Rajagopalan K.V. Enemark J.H. Rees D.C. Cell. 1997; 91: 973-983Abstract Full Text Full Text PDF PubMed Scopus (432) Google Scholar). Chicken SO is a homodimer of 52-kDa subunits, and the N- and C-terminal domains of each subunit are linked by a hinge region that is very flexible and is only weakly defined in the crystal structure (11Kisker C. Schindelin H. Pacheco A. Wehbi W.A. Garrett R.M. Rajagopalan K.V. Enemark J.H. Rees D.C. Cell. 1997; 91: 973-983Abstract Full Text Full Text PDF PubMed Scopus (432) Google Scholar). The 10-kDa N-terminal domain (residues 1–84 in the chicken SO crystal structure) contains a b5-type heme that is deeply buried but not covalently linked to the protein. The heme domain of rat SO can be removed by trypsin cleavage at the hinge region, and the remaining C-terminal molybdenum domain retains both its dimeric structure and the MPT cofactor (12Johnson J.L. Rajagopalan K.V. J. Biol. Chem. 1977; 252: 2017-2025Abstract Full Text PDF PubMed Google Scholar). In addition, the molybdenum domain has the ability to oxidize sulfite in the presence of a nonspecific electron acceptor such as ferricyanide (12Johnson J.L. Rajagopalan K.V. J. Biol. Chem. 1977; 252: 2017-2025Abstract Full Text PDF PubMed Google Scholar). The 42-kDa C-terminal domain of chicken SO consists of two sub-domains: the MPT-binding region (residues 96–323 of chicken SO) and the dimerization interface (residues 324–466).The overall reaction catalyzed by SO is summarized below, and the complete reaction cycle is shown in Fig. 1. SO32-+H2O+2(cytc)ox→SO42-+2(cytc)red+2H+Reaction 1 In the reductive half of the reaction cycle, sulfite binds at the MoVI center and is oxidized to sulfate, generating a transient two-electron reduced MoIVFeIII species. In the first intramolecular electron transfer (IET), one of the two reducing equivalents generated by sulfite oxidation is transferred from MoIV to the b5-type heme in the N terminus of SO, yielding a MoVFeII species that can be detected using EPR spectroscopy (13Cohen H.J. Fridovich I. Rajagopalan K.V. J. Biol. Chem. 1971; 246: 374-382Abstract Full Text PDF PubMed Google Scholar). In the oxidative half of the reaction cycle, one electron is transferred from the heme FeII to the terminal electron acceptor, cytochrome cox. After a second IET from MoV to FeIII, which produces the MoVIFeII state of the enzyme, the fully oxidized species is regenerated by electron transfer from FeII to a second molecule of cytochrome cox. The second IET step (from MoV to FeIII) has been extensively studied in reverse after reduction of the heme by one electron using flash photolysis (14Feng C. Kedia R.V. Hazzard J.T. Hurley J.K. Tollin G. Enemark J.H. Biochemistry. 2002; 41: 5816-5821Crossref PubMed Scopus (107) Google Scholar, 15Feng C. Wilson H.L. Hurley J.K. Hazzard J.T. Tollin G. Rajagopalan K.V. Enemark J.H. J. Biol. Chem. 2003; 278: 2913-2920Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar, 16Sullivan Jr., E.P. Hazzard J.T. Tollin G. Enemark J.H. Biochemistry. 1993; 32: 12465-12470Crossref PubMed Scopus (57) Google Scholar, 17Pacheco A. Hazzard J.T. Tollin G. Enemark J.H. J. Biol. Inorg. Chem. 1999; 4: 390-401Crossref PubMed Scopus (89) Google Scholar, 18Sullivan Jr., E.P. Hazzard J.T. Tollin G. Enemark J.H. J. Am. Chem. Soc. 1992; 114: 9662-9663Crossref Scopus (35) Google Scholar). In addition, the reductive and oxidative half-reactions of chicken SO have been analyzed using stopped-flow analysis (19Brody M.S. Hille R. Biochemistry. 1999; 38: 6668-6677Crossref PubMed Scopus (110) Google Scholar, 20Brody M.S. Hille R. Biochim. Biophys. Acta. 1995; 1253: 133-135Crossref PubMed Scopus (33) Google Scholar), which demonstrated that the rate of the reductive half-reaction limited the overall rate at pH values over 7.0.In the crystal structure of chicken SO, the molybdenum atom is pentacoordinated in a square pyramidal geometry with three sulfur and two terminal oxo ligands. Two of the sulfur atoms are from the dithiolene group of MPT, and the third is from the side chain of Cys185 (Cys207 in human SO). The dithiolene sulfur atoms of the MPT cofactor are essential for coordinating the molybdenum atom during oxo transfer from water to the sulfite molecule (4Schindelin H. Kisker C. Rajagopalan K.V. Adv. Protein Chem. 2001; 58: 47-94Crossref PubMed Scopus (66) Google Scholar). The importance of the Cys207 residue has been demonstrated by site-directed mutagenesis (21Garrett R.M. Rajagopalan K.V. J. Biol. Chem. 1996; 271: 7387-7391Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar) and EPR studies (22George G.N. Garrett R.M. Prince R.C. Rajagopalan K.V. J. Am. Chem. Soc. 1996; 118: 8588-8592Crossref Scopus (136) Google Scholar, 23Astashkin A.V. Raitsimring A.M. Feng C. Johnson J.L. Rajagopalan K.V. Enemark J.H. J. Am. Chem. Soc. 2002; 124: 6109-6118Crossref PubMed Scopus (41) Google Scholar) on human SO where a Cys207 to Ser mutation resulted in a trioxo molybdenum center with severely reduced activity. A combination of spectroscopic techniques including extended x-ray absorption fine structure (EXAFS) and resonance Raman have shown that in the oxidized state, additional coordination to the MoVI atom is provided by two terminal (equatorial and axial) oxo groups with bond lengths of 1.71 Å (22George G.N. Garrett R.M. Prince R.C. Rajagopalan K.V. J. Am. Chem. Soc. 1996; 118: 8588-8592Crossref Scopus (136) Google Scholar, 24George G.N. Kipke C.A. Prince R.C. Sunde R.A. Enemark J.H. Cramer S.P. Biochemistry. 1989; 28: 5075-5080Crossref PubMed Scopus (130) Google Scholar, 25Garton S.D. Garrett R.M. Rajagopalan K.V. Johnson M.K. J. Am. Chem. Soc. 1997; 119: 2590-2591Crossref Scopus (60) Google Scholar). Although chicken SO was purified in the fully oxidized MoVI/FeIII state, the equatorial oxo ligand in the crystal structure was assigned as a water/hydroxo molecule based onaMo–O bond length distance of 2.3 Å. In addition, a mixture of sulfite and sulfate appeared to be present in the active site near the equatorial water/hydroxo ligand, indicating that the structure obtained was that of reduced enzyme (either MoV or MoIV) with bound product. Redox cycling studies have shown that only one oxo ligand is exchanged during the reaction cycle (25Garton S.D. Garrett R.M. Rajagopalan K.V. Johnson M.K. J. Am. Chem. Soc. 1997; 119: 2590-2591Crossref Scopus (60) Google Scholar), and these studies combined with the crystal structure indicate that it is probably the equatorial oxo/hydroxo ligand, and not the apical ligand, that is catalytically active. In light of the rapid IET rates (>1000 s-1) observed in this enzyme (14Feng C. Kedia R.V. Hazzard J.T. Hurley J.K. Tollin G. Enemark J.H. Biochemistry. 2002; 41: 5816-5821Crossref PubMed Scopus (107) Google Scholar, 15Feng C. Wilson H.L. Hurley J.K. Hazzard J.T. Tollin G. Rajagopalan K.V. Enemark J.H. J. Biol. Chem. 2003; 278: 2913-2920Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar, 16Sullivan Jr., E.P. Hazzard J.T. Tollin G. Enemark J.H. Biochemistry. 1993; 32: 12465-12470Crossref PubMed Scopus (57) Google Scholar, 17Pacheco A. Hazzard J.T. Tollin G. Enemark J.H. J. Biol. Inorg. Chem. 1999; 4: 390-401Crossref PubMed Scopus (89) Google Scholar), one surprising finding from the crystal structure was the large intramolecular distance of 32 Å between the molybdenum and iron centers. The most likely explanation for the rapid IET rates is that despite the fairly extensive network of hydrogen bonds seen between the two domains in the crystal structure, the two domains adopt a conformation closer to each other in solution. In fact, IET rates decrease with increasing viscosity of the solution, indicating that domain movement is essential for efficient electron transfer between the heme and molybdenum domains of SO (14Feng C. Kedia R.V. Hazzard J.T. Hurley J.K. Tollin G. Enemark J.H. Biochemistry. 2002; 41: 5816-5821Crossref PubMed Scopus (107) Google Scholar, 15Feng C. Wilson H.L. Hurley J.K. Hazzard J.T. Tollin G. Rajagopalan K.V. Enemark J.H. J. Biol. Chem. 2003; 278: 2913-2920Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar). In addition, a recent electron-electron double resonance spectroscopy study analyzing the one-electron reduced paramagnetic (MoVFeIII) form of SO detected a range of Mo–Fe distances in solution, further demonstrating that the protein exists in multiple conformations (26Codd R. Astashkin A.V. Pacheco A. Raitsimring A.M. Enemark J.H. J. Biol. Inorg. Chem. 2002; 7: 338-350Crossref PubMed Scopus (48) Google Scholar).Based on the crystal structure, the residues forming the substrate-binding pocket of chicken SO are three arginines at positions 138, 190, and 450 as well as Tyr322 and Trp204. All five of these residues are conserved in all mammalian SO proteins isolated to date. The importance of Arg138 (Arg160 in human SO) was demonstrated by the identification of the R160Q variant in a human patient with severe SO deficiency (27Garrett R.M. Johnson J.L. Graf T.N. Feigenbaum A. Rajagopalan K.V. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 6394-6398Crossref PubMed Scopus (122) Google Scholar). Whereas mutations of the other substrate binding pocket residues have yet to be observed in human patients, Tyr322 (Tyr343 in human SO) was proposed to be important in labilizing the oxo group of MoVI to allow oxygen transfer to sulfite based on the proximity of this residue to the equatorial oxo/hydroxo group in the crystal structure (11Kisker C. Schindelin H. Pacheco A. Wehbi W.A. Garrett R.M. Rajagopalan K.V. Enemark J.H. Rees D.C. Cell. 1997; 91: 973-983Abstract Full Text Full Text PDF PubMed Scopus (432) Google Scholar). Rapid kinetic assays of the reductive half-reaction of chicken SO (19Brody M.S. Hille R. Biochemistry. 1999; 38: 6668-6677Crossref PubMed Scopus (110) Google Scholar) indicated a role for unprotonated Tyr322 in binding the substrate, based on a pKa value of 9.3 for the specificity constant (kred/Kd). Furthermore, this residue has been proposed to serve an additional role as a proton shuttle between water (or OH-) in the coupled electron-proton transfer (CEPT) mechanism during reduction and reoxidation of the molybdenum center (23Astashkin A.V. Raitsimring A.M. Feng C. Johnson J.L. Rajagopalan K.V. Enemark J.H. J. Am. Chem. Soc. 2002; 124: 6109-6118Crossref PubMed Scopus (41) Google Scholar). A recent study of human SO using flash photolysis has directly demonstrated the importance of Tyr343 in facilitating IET at low pH between the molybdenum and heme metal centers (15Feng C. Wilson H.L. Hurley J.K. Hazzard J.T. Tollin G. Rajagopalan K.V. Enemark J.H. J. Biol. Chem. 2003; 278: 2913-2920Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar). The studies described here were designed to extend our understanding of the catalytic mechanism of this essential protein through detailed kinetic analyses of wild type and the Y343F variant of human SO.The initial two-electron reduction of the MoVI center by sulfite is virtually impossible to measure spectroscopically in the full-length SO protein due to the presence of the heme cofactor. However, if the 10-kDa N-terminal heme domain of SO is removed, the remaining C-terminal molybdenum domain retains both its dimeric structure and the ability to oxidize sulfite when ferricyanide rather than cyt c is used as the terminal electron acceptor (12Johnson J.L. Rajagopalan K.V. J. Biol. Chem. 1977; 252: 2017-2025Abstract Full Text PDF PubMed Google Scholar). Recombinant production of this domain in the absence of the interfering heme chromophore has allowed spectroscopic analyses to be performed in which the absorption spectrum of the molybdenum center could be directly monitored (9Garrett R.M. Rajagopalan K.V. J. Biol. Chem. 1994; 269: 272-276Abstract Full Text PDF PubMed Google Scholar, 21Garrett R.M. Rajagopalan K.V. J. Biol. Chem. 1996; 271: 7387-7391Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar, 28Temple C.A. Graf T.N. Rajagopalan K.V. Arch. Biochem. Biophys. 2000; 383: 281-287Crossref PubMed Scopus (108) Google Scholar). In this study, the molybdenum domain of both wild-type human SO and the Y343F variant were recombinantly expressed and purified. Kinetic analyses, including steady-state and stopped-flow assays on both wild-type and the Y343F molybdenum domain, were performed in which the rate of the reduction of the molybdenum center by sulfite was directly measured for the first time. The results obtained from stopped-flow assays on the molybdenum center demonstrate that the initial reduction of the molybdenum center by sulfite is not the rate-limiting step in overall catalysis at either high or low pH in either the wild-type protein or the Y343F variant. Taken together, the data presented in this study corroborate the proposed role of Tyr343 in substrate binding as well as catalysis in this essential human protein.EXPERIMENTAL PROCEDURESThe Y343F mutation was introduced into the wild-type human SO-containing plasmid construct pTG918 (28Temple C.A. Graf T.N. Rajagopalan K.V. Arch. Biochem. Biophys. 2000; 383: 281-287Crossref PubMed Scopus (108) Google Scholar) using the Transformer site-directed mutagenesis kit (Clontech) with the mutagenic primer (GGCGGGATTTCAAAGGCTTCTC) as described previously (15Feng C. Wilson H.L. Hurley J.K. Hazzard J.T. Tollin G. Rajagopalan K.V. Enemark J.H. J. Biol. Chem. 2003; 278: 2913-2920Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar). The Y343F molybdenum domain construct was generated by cleavage of pTG918 Y343F with the restriction enzymes NdeI and HindIII. The resulting 640-bp fragment spanning the Y343F mutation was then ligated into the similarly digested wild-type molybdenum domain construct pTG818 (28Temple C.A. Graf T.N. Rajagopalan K.V. Arch. Biochem. Biophys. 2000; 383: 281-287Crossref PubMed Scopus (108) Google Scholar). All constructs were verified by sequence analysis performed at the Duke University DNA analysis facility.Both recombinant wild-type and Y343F human SO were expressed and purified as previously described (21Garrett R.M. Rajagopalan K.V. J. Biol. Chem. 1996; 271: 7387-7391Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar, 28Temple C.A. Graf T.N. Rajagopalan K.V. Arch. Biochem. Biophys. 2000; 383: 281-287Crossref PubMed Scopus (108) Google Scholar) with the following modifications. After the phenyl-Sepharose column, fractions exhibiting an A413/A280 ratio greater than 0.89 were pooled and further purified using a Zorbax GF-250 high performance liquid chromatography gel filtration column (Agilent Technologies) in 250 mm KPO4 buffer at pH 7.80. Fractions exhibiting an A413/A280 ratio ≥ 0.96 were then pooled and used in the experiments described in this study. The His6-tagged molybdenum domains of both the wild-type and Y343F variant were purified as previously described (28Temple C.A. Graf T.N. Rajagopalan K.V. Arch. Biochem. Biophys. 2000; 383: 281-287Crossref PubMed Scopus (108) Google Scholar). The molybdenum content of purified proteins was determined using a PerkinElmer Zeeman/3030 atomic absorption spectrometer as previously described (21Garrett R.M. Rajagopalan K.V. J. Biol. Chem. 1996; 271: 7387-7391Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar, 29Johnson J.L. Methods Enzymol. 1988; 158: 371-382Crossref PubMed Scopus (13) Google Scholar).Steady-state kinetic assays were performed aerobically at 25 °C using a 1.0-cm path length cuvette in a Shimadzu UV-1601PC spectrophotometer. Assays were carried out in 20 or 100 mm buffers adjusted to the desired pH with acetic acid to minimize anion inhibition of SO (19Brody M.S. Hille R. Biochemistry. 1999; 38: 6668-6677Crossref PubMed Scopus (110) Google Scholar, 30Kessler D.L. Rajagopalan K.V. Biochim. Biophys. Acta. 1974; 370: 389-398Crossref PubMed Scopus (37) Google Scholar). The buffers used were as follows: Bis-Tris (pH 6.0–6.5), Bis-Tris propane (pH 6.75–7.5), Tris (pH 7.5–8.75), and glycine (pH 8.75–10.0). The glycine buffers were adjusted to the desired pH using NaOH. The steady-state pH profile was performed using 15–50 μm horse heart cyt c (Sigma), 0.50–2.5 μg/ml SO, and varying concentrations of sulfite in a final assay volume of 1 ml. The concentration of SO was determined from the A413 using an extinction coefficient of 113 mm-1 cm-1. The reduction of cyt c was monitored at 550 nm using an extinction coefficient of 19 mm-1 cm-1.Steady-state assays using ferricyanide as the electron acceptor were performed using 50 mm buffers, 40 μm ferricyanide, 1.5–5.0 μg/ml SO, and varying concentrations of sulfite in a final assay volume of 1 ml. The reduction of ferricyanide was monitored at 420 nm, and enzyme activity was reported in units/mg, where a unit is equal to a change of 1.0 absorbance units/min at 420 nm.Rapid reaction kinetic studies were performed using an SX.18MV stopped-flow reaction analyzer (Applied Photophysics Ltd.). The dead time of the instrument was determined to be <1.7 ms with a path length of 10 mm. To prepare the instrument for anaerobic operation, the flow tubing and valve lines were incubated with 250 mm sodium dithionite for several hours and then thoroughly rinsed with O2-free water. The sample-handling unit of the stopped-flow apparatus was fitted with an anaerobic accessory that enclosed the drive syringe plungers and was flushed continuously with nitrogen gas. The temperature for the reaction was maintained at either 10 or 25 °C using a circulating water bath connected to the thermostat bath housing the drive syringes. All buffers were made anaerobic by bubbling with argon gas for at least 30 min prior to use, and solutions of human SO were prepared by diluting <100 μl of a concentrated stock of the enzyme into a final volume of ∼5 ml of anaerobic buffer. All dilutions were performed in an anaerobic chamber (Coy Laboratory Products Inc.), and 5-ml gas-tight syringes (Hamilton Co.) were used to load samples into the stopped-flow spectrophotometer. For full-length wild-type human SO and the Y343F variant, rapid kinetic assays of the reductive half-reaction were performed anaerobically at 25 °C using 0.3–0.8 μm protein. Reduction of the b5-type heme of SO was directly monitored by measuring the extinction change of the Soret peak at 415, 426, or 430 nm. Similar rate constants were obtained when the reaction was monitored by measuring the α peak at 560 nm. For the molybdenum domain of wild-type and Y343F human SO, the greatest spectral change between the reduced and oxidized species was seen between 350 and 365 nm. Stopped-flow assays were performed anaerobically at 10 °C using 15–18 μm protein, and the extinction change was monitored at 355 or 360 nm. For both full-length and molybdenum domain assays, the kobs of each individual reaction was obtained by fitting individual kinetic traces to single exponential curves using a nonlinear least-squares Levenberg-Marquardt algorithm.The steady-state reaction parameters kcat and Km were obtained by a direct fit of the concentration dependence to the Michaelis-Menten equation. The maximal rate parameter kred and the Kdsulfite for the reductive half-reactions were obtained by fitting the kobs at varying sulfite concentrations to a hyperbolic curve (Equation 1). kobs=kred[S]/(Kd+[S])(Eq. 1) The limiting rate parameters (kcat or kred for steady state and stopped-flow assays, respectively) were plotted as a function of pH, and the pKa values were obtained using Equation 2 for bell-shaped double ionization curves and Equation 3 for sigmoidal single ionization curves as described previously by Brody and Hille (19Brody M.S. Hille R. Biochemistry. 1999; 38: 6668-6677Crossref PubMed Scopus (110) Google Scholar). kcat/Km=Tmax/(1+10(pKa1-pH)+10(pH-pKd2))(Eq. 2) kred/Kd=((AEH(10(-pH)))+(AE(10(-pKa)))]/[(10(-pH))+(10(-pKa)))(Eq. 3) In Equation 2, Tmax represents the theoretical maximal value of the kinetic parameter where it is assumed that only the middle ionization state (EH for the double equilibrium E = EH = EH2) is catalytically active. In Equation 3, AEH and AE represent the theoretical catalytic activity of the protonated and unprotonated forms, respectively, of the ionization group.The atomic coordinates of the crystal structure of chicken SO (11Kisker C. Schindelin H. Pacheco A. Wehbi W.A. Garrett R.M. Rajagopalan K.V. Enemark J.H. Rees D.C. Cell. 1997; 91: 973-983Abstract Full Text Full Text PDF PubMed Scopus (432) Google Scholar) in the Molecular Modeling Data base (available on the World Wide Web at www.ncbi.nlm.nih.gov:80/Structure/MMDB/mmdb.shtml) under MMDB 7838 were used to create Fig. 6 using the PyMOL Molecular Graphics System (W. L. DeLano; available on the World Wide Web at www.pymol.org).Fig. 6Structure of the chicken SO substrate-binding site. The positions of Tyr322 and Arg138 in the substrate-binding site of chicken SO are indicated. The MPT cofactor and the sulfate molecule bound in the crystal structure are shown in a ball-and-stick representation. Hydrogen bonds are indicated by dotted gray lines. The molybdenum atom is shown in green. The PyMOL Molecular Graphics Program (W. L. DeLano; available on the World Wide Web at www.pymol.org) was used to create this figure after obtaining the atomic coordinates of the crystal structure of chicken SO (11Kisker C. Schindelin H. Pacheco A. Wehbi W.A. Garrett R.M. Rajagopalan K.V. Enemark J.H. Rees D.C. Cell. 1997; 91: 973-983Abstract Full Text Full Text PDF PubMed Scopus (432) Google Scholar) deposited in the Molecular Modeling Database under MMDB 7838.View Large Image Figure ViewerDownload Hi-res image Download (PPT)RESULTSSteady-state Kinetic Assays of Wild-type and Y343F Human SO—Based on the crystal structure of chicken SO, Tyr322 was proposed to have an important role in mediating the substrate specificity and catalytic activity of SO (11Kisker C. Schindelin H. Pacheco A. Wehbi W.A. Garrett R.M. Rajagopalan K.V. Enemark J.H. Rees D.C. Cell. 1997; 91: 973-983Abstract Full Text Full Text PDF PubMed Scopus (432) Google Scholar). Additionally, a previous kinetic study of native chicken SO by Brody and Hille proposed a role for this residue in binding of the substrate (19Brody M.S. Hille R. Biochemistry. 1999; 38: 6668-6677Crossref PubMed Scopus (110) Google Scholar). Since Tyr343 is the human equivalent of the chicken Tyr322, the Y343F variant of human SO was generated and purified from recombinant Escherichia coli. The protein behaved similarly to wild-type human SO throughout all purification steps, and the Mo/heme ratio as measured using atomic absorption spectroscopy ranged from 0.78 to 0.85 in all protein preparations used in this study.Initial steady-state assays were performed on human SO and the Y343F variant using cyt c as the terminal electron acceptor over the pH range 7–10 for both wild-type and Y343F SO (Table I). The activity of the Y343F variant was impaired across the entire pH range, with a more significant loss in activity observed at low pH val
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