Biophysical and Structural Analysis of a Novel Heme b Iron Ligation in the Flavocytochrome Cellobiose Dehydrogenase
2003; Elsevier BV; Volume: 278; Issue: 35 Linguagem: Inglês
10.1074/jbc.m302653200
ISSN1083-351X
AutoresFrederik A.J. Rotsaert, B.M. Hallberg, Simon de Vries, Pierre Moënne‐Loccoz, Christina Divne, V. Renganathan, Michael H. Gold,
Tópico(s)Enzyme Structure and Function
ResumoThe fungal extracellular flavocytochrome cellobiose dehydrogenase (CDH) participates in lignocellulose degradation. The enzyme has a cytochrome domain connected to a flavin-binding domain by a peptide linker. The cytochrome domain contains a 6-coordinate low spin b-type heme with unusual iron ligands and coordination geometry. Wild type CDH is only the second example of a b-type heme with Met-His ligation, and it is the first example of a Met-His ligation of heme b where the ligands are arranged in a nearly perpendicular orientation. To investigate the ligation further, Met65 was replaced with a histidine to create a bis-histidyl ligated iron typical of b-type cytochromes. The variant is expressed as a stable 90-kDa protein that retains the flavin domain catalytic reactivity. However, the ability of the mutant to reduce external one-electron acceptors such as cytochrome c is impaired. Electrochemical measurements demonstrate a decrease in the redox midpoint potential of the heme by 210 mV. In contrast to the wild type enzyme, the ferric state of the protoheme displays a mixed low spin/high spin state at room temperature and low spin character at 90 K, as determined by resonance Raman spectroscopy. The wild type cytochrome does not bind CO, but the ferrous state of the variant forms a CO complex, although the association rate is very low. The crystal structure of the M65H cytochrome domain has been determined at 1.9 Å resolution. The variant structure confirms a bis-histidyl ligation but reveals unusual features. As for the wild type enzyme, the ligands have a nearly perpendicular arrangement. Furthermore, the iron is bound by imidazole Nδ1 and Nϵ2 nitrogen atoms, rather than the typical Nϵ2/Nϵ2 coordination encountered in bis-histidyl ligated heme proteins. To our knowledge, this is the first example of a bis-histidyl Nδ1/Nϵ2-coordinated protoporphyrin IX iron. The fungal extracellular flavocytochrome cellobiose dehydrogenase (CDH) participates in lignocellulose degradation. The enzyme has a cytochrome domain connected to a flavin-binding domain by a peptide linker. The cytochrome domain contains a 6-coordinate low spin b-type heme with unusual iron ligands and coordination geometry. Wild type CDH is only the second example of a b-type heme with Met-His ligation, and it is the first example of a Met-His ligation of heme b where the ligands are arranged in a nearly perpendicular orientation. To investigate the ligation further, Met65 was replaced with a histidine to create a bis-histidyl ligated iron typical of b-type cytochromes. The variant is expressed as a stable 90-kDa protein that retains the flavin domain catalytic reactivity. However, the ability of the mutant to reduce external one-electron acceptors such as cytochrome c is impaired. Electrochemical measurements demonstrate a decrease in the redox midpoint potential of the heme by 210 mV. In contrast to the wild type enzyme, the ferric state of the protoheme displays a mixed low spin/high spin state at room temperature and low spin character at 90 K, as determined by resonance Raman spectroscopy. The wild type cytochrome does not bind CO, but the ferrous state of the variant forms a CO complex, although the association rate is very low. The crystal structure of the M65H cytochrome domain has been determined at 1.9 Å resolution. The variant structure confirms a bis-histidyl ligation but reveals unusual features. As for the wild type enzyme, the ligands have a nearly perpendicular arrangement. Furthermore, the iron is bound by imidazole Nδ1 and Nϵ2 nitrogen atoms, rather than the typical Nϵ2/Nϵ2 coordination encountered in bis-histidyl ligated heme proteins. To our knowledge, this is the first example of a bis-histidyl Nδ1/Nϵ2-coordinated protoporphyrin IX iron. Cellobiose dehydrogenases (CDHs) 1The abbreviations used are: CDH, cellobiose dehydrogenase; 6cLS, 6-coordinate low spin; cyt c, cytochrome c; CYTM65H, M65H cytochrome domain; DCPIP, 2,6-dichlorophenol-indophenol; ET, electron transfer; HCHN, high carbon-high nitrogen; HS, high spin; LS, low spin; rCDH, recombinant wild type CDH; RR, resonance Raman; DCPIP, 2,6-dichlorophenol-indophenol.1The abbreviations used are: CDH, cellobiose dehydrogenase; 6cLS, 6-coordinate low spin; cyt c, cytochrome c; CYTM65H, M65H cytochrome domain; DCPIP, 2,6-dichlorophenol-indophenol; ET, electron transfer; HCHN, high carbon-high nitrogen; HS, high spin; LS, low spin; rCDH, recombinant wild type CDH; RR, resonance Raman; DCPIP, 2,6-dichlorophenol-indophenol. are extracellular fungal flavocytochromes with a role in the biodegradation of lignocellulose (1Henriksson G. Johansson G. Pettersson G. J. Biotechnol. 2000; 78: 93-113Crossref PubMed Scopus (357) Google Scholar). The CDH gene from the white rot fungus Phanerochaete chrysosporium has been cloned and sequenced (2Li B. Nagalla S.R. Renganathan V. Appl. Environ. Microbiol. 1996; 62: 1329-1335Crossref PubMed Google Scholar, 3Raices M. Paifer E. Cremata J. Montesino R. Stahlberg J. Divne C. Szabo Istvan J. Henriksson G. Johansson G. Pettersson G. FEBS Lett. 1995; 369: 233-238Crossref PubMed Scopus (56) Google Scholar), revealing a full-length protein of 755 amino acids partitioned into a cytochrome domain (residues 1–190) and a flavodehydrogenase domain (residues 216–755) connected by a 25-residue peptide linker. A flavin adenine dinucleotide cofactor is bound to the flavoprotein domain, whereas the cytochrome domain contains a 6-coordinate low spin (6cLS) Fe-protoporphyrin IX (4Cox M.C. Rogers M.S. Cheesman M. Jones G.D. Thomson A.J. Wilson M.T. Moore G.R. FEBS Lett. 1992; 307: 233-236Crossref PubMed Scopus (30) Google Scholar, 5Cohen J.D. Bao W. Renganathan V. Subramaniam S.S. Loehr T.M. Arch. Biochem. Biophys. 1997; 341: 321-328Crossref PubMed Scopus (14) Google Scholar). In the reductive half-reaction, the flavodehydrogenase domain catalyzes the oxidation of cellobiose to yield cellobiono-1,5-lactone (6Higham C.W. Gordon-Smith D. Dempsey C.E. Wood P.M. FEBS Lett. 1994; 351: 128-132Crossref PubMed Scopus (42) Google Scholar), with the concomitant reduction of flavin adenine dinucleotide. During the ensuing oxidative half-reaction, the flavin is reoxidized by an electron acceptor, either directly for two-electron acceptors such as 2,6-dichlorophenol-indophenol (DCPIP) or via the cytochrome domain for one-electron acceptors, such as cytochrome c (cyt c).The 1.9 Å resolution crystal structure of the wild type P. chrysosporium CDH cytochrome domain has been reported elsewhere (7Hallberg B.M. Bergfors T. Backbro K. Pettersson G. Herriksson G. Divne C. Structure. 2000; 8: 79-88Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar). The heme-binding module features an unusual fold among cytochromes: an immunoglobulin-like β-sandwich consisting of a five-stranded and a six-stranded β-sheet. The protoheme group is bound in a hydrophobic pocket at one face of the β-core with one heme edge exposed to solvent. Three loops protrude from the β-sheet and wedge the b-type heme. The packing of the heme pocket formed by various nonpolar residues is tight, leaving little space for exogenous molecules. The crystal structure (7Hallberg B.M. Bergfors T. Backbro K. Pettersson G. Herriksson G. Divne C. Structure. 2000; 8: 79-88Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar) confirmed earlier spectroscopic predictions (4Cox M.C. Rogers M.S. Cheesman M. Jones G.D. Thomson A.J. Wilson M.T. Moore G.R. FEBS Lett. 1992; 307: 233-236Crossref PubMed Scopus (30) Google Scholar), that the heme iron is ligated by a methionine and histidine with an unusual, nearly perpendicular arrangement (∼100°) of the two planes defined by the methionine thioether group and the His163 imidazole ring. The distances of the iron-nitrogen and iron-sulfur bonds, 2.0 and 2.3 Å, respectively, are typical of those observed in c-type cytochromes with Met-His iron ligation.Results from site-directed mutagenesis of the two protoheme-iron ligands confirmed their importance (8Rotsaert F.A. Li B. Renganathan V. Gold M.H. Arch. Biochem. Biophys. 2001; 390: 206-214Crossref PubMed Scopus (13) Google Scholar). Substitution of either residue with an alanine demonstrated that the Met-His coordination is essential for heme reactivity, i.e. the electron transfer (ET) to one-electron acceptors. In addition, the loss of an axial protein ligand rendered the cytochrome domain highly susceptible to degradation. Indeed, similar mutant studies in other b-type cytochromes reveal a weaker binding (9Miles C.S. Manson F.D. Reid G.A. Chapman S.K. Biochim. Biophys. Acta. 1993; 1202: 82-86Crossref PubMed Scopus (11) Google Scholar) or nonincorporation of the heme (10Hampsey D.M. Das G. Sherman F. FEBS Lett. 1988; 231: 275-283Crossref PubMed Scopus (57) Google Scholar, 11Davis C.A. Dhawan I.K. Johnson M.K. Barber M.J. Arch. Biochem. Biophys. 2002; 400: 63-75Crossref PubMed Scopus (16) Google Scholar, 12Beck von Bodman S. Schuler M.A. Jollie D.R. Sligar S.G. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 9443-9447Crossref PubMed Scopus (197) Google Scholar). Loss of the protoheme in the alanine variants of CDH may lead to unfolding of the cytochrome domain, rendering it more susceptible to proteolytic cleavage. In contrast, in c-type cytochromes, replacing the axially ligated methionine with a histidine produced a stable protein with some properties similar to the wild type (13Aubert C. Guerlesquin F. Bianco P. Leroy G. Tron P. Stetter K.O. Bruschi M. Biochemistry. 2001; 40: 13690-13698Crossref PubMed Scopus (18) Google Scholar, 14Darrouzet E. Mandaci S. Li J. Qin H. Knaff D.B. Daldal F. Biochemistry. 1999; 38: 7908-7917Crossref PubMed Scopus (23) Google Scholar, 15Miller G.T. Zhang B. Hardman J.K. Timkovich R. Biochemistry. 2000; 39: 9010-9017Crossref PubMed Scopus (28) Google Scholar, 16Raphael A.L. Gray H.B. Proteins. 1989; 6: 338-340Crossref PubMed Scopus (87) Google Scholar). Speculation about the coordination geometry to the protoheme in these variants has been advanced (14Darrouzet E. Mandaci S. Li J. Qin H. Knaff D.B. Daldal F. Biochemistry. 1999; 38: 7908-7917Crossref PubMed Scopus (23) Google Scholar, 16Raphael A.L. Gray H.B. Proteins. 1989; 6: 338-340Crossref PubMed Scopus (87) Google Scholar), but no structural studies have been reported. Herein, we report the results from site-directed mutagenesis, kinetic, electrochemical, spectroscopic, and crystallographic studies on the M65H variant of P. chrysosporium CDH.EXPERIMENTAL PROCEDURESOrganism—Growth and maintenance of the auxotrophic strain OGC316-7 (Ura 11) and prototrophic transformants were as described previously (17Akileswaran L. Alic M. Clark E.K. Hornick J.L. Gold M.H. Curr. Genet. 1993; 23: 351-356Crossref PubMed Scopus (29) Google Scholar, 18Alic M. Clark E.K. Kornegay J.R. Gold M.H. Curr. Genet. 1990; 17: 305-311Crossref Scopus (47) Google Scholar). Escherichia coli DH5α was used for subcloning plasmids.Construction of the Mutant Plasmid pM65H—The M65H site-directed mutation was introduced into pUGC1 using the Transform™ site-directed mutagenesis kit (Clontech Laboratories, Palo Alto, CA) (8Rotsaert F.A. Li B. Renganathan V. Gold M.H. Arch. Biochem. Biophys. 2001; 390: 206-214Crossref PubMed Scopus (13) Google Scholar). The mutant primer converted the ATG codon (Met) to the CAC codon (His). The mutant plasmid pM65H was isolated, and the mutation was confirmed by sequencing.Transformation of P. chrysosporium with pM65H—Protoplasts of P. chrysosporium OGC316-7 (Ura11), a uracil auxotroph, were prepared as described previously (19Li B. Rotsaert F.A.J. Gold M.H. Renganathan V. Biochem. Biophys. Res. Commun. 2000; 270: 141-146Crossref PubMed Scopus (16) Google Scholar, 20Sollewijn Gelpke M.D. Mayfield-Gambill M. Cereghino G.P.L. Gold M.H. Appl. Environ. Microbiol. 1999; 65: 1670-1674Crossref PubMed Google Scholar) and transformed with EcoRI-linearized pM65H (2 μg), and potential transformants were screened for uracil prototrophy (19Li B. Rotsaert F.A.J. Gold M.H. Renganathan V. Biochem. Biophys. Res. Commun. 2000; 270: 141-146Crossref PubMed Scopus (16) Google Scholar, 20Sollewijn Gelpke M.D. Mayfield-Gambill M. Cereghino G.P.L. Gold M.H. Appl. Environ. Microbiol. 1999; 65: 1670-1674Crossref PubMed Google Scholar). Conidia from prototrophs were then cultured in high carbon high nitrogen (HCHN) stationary liquid cultures with glucose as the sole carbon source (8Rotsaert F.A. Li B. Renganathan V. Gold M.H. Arch. Biochem. Biophys. 2001; 390: 206-214Crossref PubMed Scopus (13) Google Scholar, 19Li B. Rotsaert F.A.J. Gold M.H. Renganathan V. Biochem. Biophys. Res. Commun. 2000; 270: 141-146Crossref PubMed Scopus (16) Google Scholar) and assayed for extracellular CDH activity using both the cyt c and DCPIP assays (8Rotsaert F.A. Li B. Renganathan V. Gold M.H. Arch. Biochem. Biophys. 2001; 390: 206-214Crossref PubMed Scopus (13) Google Scholar, 19Li B. Rotsaert F.A.J. Gold M.H. Renganathan V. Biochem. Biophys. Res. Commun. 2000; 270: 141-146Crossref PubMed Scopus (16) Google Scholar). The transformant exhibiting the highest activity was purified by isolating single basidiospores as described elsewhere (21Alic M. Kornegay J.R. Pribnow D. Gold M.H. Appl. Environ. Microbiol. 1989; 55: 406-411Crossref PubMed Google Scholar, 22Alic M. Mayfield M.B. Akileswaran L. Gold M.H. Curr. Genet. 1991; 19: 491-494Crossref Scopus (34) Google Scholar), and progeny were rescreened for CDH activity in liquid cultures.Production and Purification of the M65H Variant—The M65H strain was grown for 7 days at 37 °C from conidial inocula in HCHN stationary liquid cultures with glucose as the sole carbon source. The extracellular fluid from 7-day-old cultures was concentrated and dialyzed against 20 mm potassium phosphate, pH 6. Subsequently, the variant protein was purified by cellulose affinity chromatography, gel filtration (Sephacryl S200 HR), and fast protein liquid chromatography using a MonoQ HR5/5 anion exchanger, as described previously (8Rotsaert F.A. Li B. Renganathan V. Gold M.H. Arch. Biochem. Biophys. 2001; 390: 206-214Crossref PubMed Scopus (13) Google Scholar).Preparation of Cytochrome Domains—The cytochrome domains of recombinant wild type CDH (rCDH) and M65H (CYTM65H) were obtained by limited proteolysis with papain (5Cohen J.D. Bao W. Renganathan V. Subramaniam S.S. Loehr T.M. Arch. Biochem. Biophys. 1997; 341: 321-328Crossref PubMed Scopus (14) Google Scholar, 23Henriksson G. Pettersson G. Johansson G. Ruiz A. Uzcategui E. Eur. J. Biochem. 1991; 196: 101-106Crossref PubMed Scopus (140) Google Scholar) and purified by fast protein liquid chromatography using a MonoQ anion exchanger with a 0–1 m NaCl gradient in 10 mm Tris-HCl, pH 8.SDS-PAGE and Western Blot Analysis—SDS-PAGE was performed using a 12% Tris-glycine system (24Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (206048) Google Scholar) in a Miniprotean II apparatus (Bio-Rad), and the gels were stained with Coomassie Blue. Western blot analysis was performed as described previously (8Rotsaert F.A. Li B. Renganathan V. Gold M.H. Arch. Biochem. Biophys. 2001; 390: 206-214Crossref PubMed Scopus (13) Google Scholar).Estimation of Protein and Heme Content—The protein concentration was determined by the bicinchoninic acid method (25Smith P.K. Krohn R.I. Hermanson G.T. Mallia A.K. Gartner F.H. Provenzano M.D. Fujimoto E.K. Goeke N.M. Olson B.J. Klenk D.C. Anal. Biochem. 1985; 150: 76-85Crossref PubMed Scopus (18446) Google Scholar). The heme content was estimated by the pyridine hemochromogen procedure (26Berry E.A. Trumpower B.L. Anal. Biochem. 1987; 161: 1-15Crossref PubMed Scopus (737) Google Scholar).Spectroscopic Procedures—Electronic absorption spectra of rCDH and the M65H variant were recorded at room temperature with a Cary 100 spectrophotometer. The spectra were obtained in 20 mm sodium succinate, pH 4.5. The enzymes were reduced under aerobic or anaerobic conditions by addition of cellobiose (200 μm) or excess dithionite. The CO adduct of the reduced form of the M65H variant was obtained by briefly bubbling CO gas through a cellobiose- or dithionite-reduced enzyme solution under anaerobic conditions. To measure the association rate of CO, native M65H variant (∼1.5 μm) was added to an anaerobic solution of 20 mm sodium succinate, pH 4.5, containing 60–240 μm CO and >100 μm dithionite. Ligand association was followed by the change in the absorbance at 431 nm.Resonance Raman Spectroscopy—Resonance Raman spectra were measured on 15 μl of each sample sealed in a glass melting point capillary tube using a custom McPherson 2061/207 spectrograph equipped with a Princeton Instruments LN1100PB liquid N2-cooled CCD detector and Kaiser Optical Systems holographic notch filter. Excitation light was provided by an Innova 302 krypton laser (413 nm). The laser power at the sample was ∼40 mW. The plasma emission lines were removed by an Applied Photophysics prism monochromator. The data at room temperature and 90 K were collected in a back-scattering geometry with the sample capillary placed in a copper cold finger. For the 90 K experiments, the capillary was cooled by liquid nitrogen. Spectral data were processed using GRAMS/386 (Galactic Industries) and Origin (Microcal) data analysis programs. The spectra were calibrated against indene as an external standard. The frequencies are estimated to be accurate to within ±1 cm–1.Enzyme Assays and Kinetic Procedure—CDH activity was measured using either the cyt c or the DCPIP assay (8Rotsaert F.A. Li B. Renganathan V. Gold M.H. Arch. Biochem. Biophys. 2001; 390: 206-214Crossref PubMed Scopus (13) Google Scholar, 19Li B. Rotsaert F.A.J. Gold M.H. Renganathan V. Biochem. Biophys. Res. Commun. 2000; 270: 141-146Crossref PubMed Scopus (16) Google Scholar). The steady state kinetic parameters for cellobiose oxidation were determined by monitoring ferrocytochrome c formation (ϵ550 = 28 mm–1 cm–1) or DCPIP reduction (ϵ515 = 6.8 mm–1 cm–1). The assays contained a fixed level of ferricytochrome c (12.5 μm) or DCPIP (35 μm) and varying levels of cellobiose (5–200 μm) in 20 mm sodium succinate, pH 4.5. The steady state kinetics for cyt c and DCPIP reduction were determined with a fixed cellobiose concentration (200 μm) and variable cyt c and DCPIP concentrations (0.2–40 μm).Potentiometric Titration—Potentiometric titrations were carried out at room temperature in a borosilicate glass cell, similar to that described previously (27Dutton P.L. Methods Enzymol. 1978; 54: 411-435Crossref PubMed Scopus (725) Google Scholar). The potential was measured with a platinum electrode versus a REF401 calomel electrode (Radiometer). All of the values are expressed with respect to the normal hydrogen electrode. The electrodes were calibrated against a pH 7 standard solution of quinhydrone (E m = +293 mV versus normal hydrogen electrode) with a Metrohm 632 pH meter (Metrohm, Herisau, Switzerland). The redox midpoint potential was determined in 50 mm sodium succinate, pH 4.5. Redox equilibration between the protein and the electrode was achieved by the use of a mixture of dyes: phenazine methosulfate, phenazine ethosulfate, 2-hydoxy-1,4-naphthoquinone, anthraquinone-1,5-disulfonate, anthraquinone-2,6-disulfonate, anthraquinone-2-sulfonate, and/or Fe3+-EDTA. The redox titration was carried out with stirring of the buffered solution (5.5 ml), containing 5 μm enzyme, the mediator dyes (20 μm each), and 50 μm Fe3+-EDTA. Prior to the reductive titration, the solution of enzyme and mediators was flushed with argon. The solution was then allowed to reach equilibrium, and the first UV-visible spectrum was recorded with an HP 8353 Diode Array spectrophotometer (Hewlett Packard, Palo Alto, CA). The redox potential of the system was adjusted by the addition of a small volume of 10 or 100 mm dithionite via a Hamilton syringe. After equilibration (constant reading of absorbance and potential), a spectrum was recorded, the was potential noted, and an additional small volume of dithionite was added. This process was repeated until the enzyme was completely reduced. The oxidative titration was carried out by the addition of small amounts of air to the cell, followed by flushing with argon. The system was allowed to equilibrate, a spectrum was recorded, and the potential was noted. This procedure was repeated until the enzyme was completely oxidized. The redox state of CDH was determined from the size of α the band of heme b: 562 nm for rCDH and 560 nm for M65H. The absorbance at this wavelength, corrected for the absorbance at 800 nm, was plotted against the potential of the system. The graph was fitted against the Nernst equation to obtain the redox potential E m. The Nernst plot for both oxidative and reductive titration exhibited no hysteresis, confirming that the system was at equilibrium.Protein Preparation for Crystallization—The M65H variant was cleaved proteolytically with papain to yield distinct cytochrome and flavin fragments as described previously (5Cohen J.D. Bao W. Renganathan V. Subramaniam S.S. Loehr T.M. Arch. Biochem. Biophys. 1997; 341: 321-328Crossref PubMed Scopus (14) Google Scholar, 23Henriksson G. Pettersson G. Johansson G. Ruiz A. Uzcategui E. Eur. J. Biochem. 1991; 196: 101-106Crossref PubMed Scopus (140) Google Scholar). The fragments were fractionated on a MonoQ HR 5/5 anion exchanger in 20 mm Tris-HCl, pH 8.0, using a linear NaCl gradient (0–1 m), followed by refraction-ation of the samples containing CYTM65H at pH 4.2, using a linear sodium acetate gradient (50 mm to 1 m). Crystals of CYTM65H were grown at room temperature using the hanging drop, vapor diffusion method (28McPherson A. Preparation and Analysis of Protein Chrystals. John Wiley & Sons, New York1982Google Scholar). Hanging drops were prepared by mixing equal volumes of protein solution (3 mg/ml) and reservoir. The reservoir contained 30% (w/v) polyethylene glycol 4000, 5% (v/v) 2-methyl 2,4-pentanediol, 100 mm HEPES, pH 7.5, and 10 mm CaCl2. The crystals appeared as red hexagonal rods of space group P65 with cell constants a = b = 139.0 Å and c = 52.67 Å and with two molecules in the asymmetric unit.X-ray Crystallographic Data Collection and Refinement—The data were collected at 100 K using synchrotron radiation (source ID14-EH4, European Synchrotron Radiation Facility, Grenoble, France; λ = 0.9763 Å). Data reduction and scaling were carried out using MOSFLM (29Powell H.R. Acta Crystallogr. Sect. D Biol. Crystallogr. 1999; 55: 1690-1695Crossref PubMed Scopus (304) Google Scholar) and SCALA (30Evans P.R. Sawyer L. Isaacs N. Bailey S. Proceedings of CCP4 Sutdy Weekend on Data Collection and Processing. SERC Daresbury Lab., Warrington, UK1993: 114-122Google Scholar), respectively. The previously reported structure of the P. chrysosporium CDH cytochrome domain at 1.9 Å resolution (Protein Data Bank code 1D7C) (7Hallberg B.M. Bergfors T. Backbro K. Pettersson G. Herriksson G. Divne C. Structure. 2000; 8: 79-88Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar) was used as a starting model for crystallographic refinement against CYTM65H amplitudes. Initial refinement and manual model building were performed with the programs CNS (31Brunger A.T. Adams P.D. Clore G.M. DeLano W.L. Gros P. Grosse-Kunstleve R.W. Jiang J.S. Kuszewski J. Nilges M. Pannu N.S. Read R.J. Rice L.M. Simonson T. Warren G.L. Acta Crystallogr. Sect. D Biol. Crystallogr. 1998; 54: 905-921Crossref PubMed Scopus (16930) Google Scholar) and O (32Jones T.A. Zou J.Y. Cowan S.W. Kjeldgaard M. Acta Crystallogr. Sect. A. 1991; 47: 110-119Crossref PubMed Scopus (13004) Google Scholar), respectively. Final refinement was done with REF-MAC5 (33Murshudov G.N. Vagin A.A. Dodson E.J. Acta Crystallogr. Sect. D Biol. Crystallogr. 1997; 53: 240-255Crossref PubMed Scopus (13779) Google Scholar) at 1.9 Å resolution, using anisotropic scaling, hydrogens in their riding positions, and atomic displacement parameter refinement, using the "translation, liberation, screw rotation" model. The two noncrystallographically related molecules were defined as rigid bodies during translation, liberation, screw rotation refinement. All least squares planes and angles between normals and least squares planes were calculated, using the program MOLEMAN2 (34Kleywegt G.J. Acta Crystallogr. Sect. D Biol. Crystallogr. 2000; 56: 249-265Crossref PubMed Scopus (183) Google Scholar).RESULTSExpression and Purification of the M65H Variant—The M65H mutation was verified by DNA sequencing. Transformation of the Ura– strain (Ura11) with linearized pM65H resulted in the isolation of several prototrophic transformants. Each was grown in liquid HCHN culture in the presence of glucose, conditions under which endogenous wild type CDH is not expressed (2Li B. Nagalla S.R. Renganathan V. Appl. Environ. Microbiol. 1996; 62: 1329-1335Crossref PubMed Google Scholar, 19Li B. Rotsaert F.A.J. Gold M.H. Renganathan V. Biochem. Biophys. Res. Commun. 2000; 270: 141-146Crossref PubMed Scopus (16) Google Scholar). The extracellular medium was monitored for CDH activity, using the cyt c and DCPIP reduction assays. Several transformants exhibited significant DCPIP reduction activity, but none efficiently reduced cyt c. The transformant exhibiting the highest DCPIP activity was purified by fruiting and isolating single basidiospore-derived colonies (21Alic M. Kornegay J.R. Pribnow D. Gold M.H. Appl. Environ. Microbiol. 1989; 55: 406-411Crossref PubMed Google Scholar, 22Alic M. Mayfield M.B. Akileswaran L. Gold M.H. Curr. Genet. 1991; 19: 491-494Crossref Scopus (34) Google Scholar). The purified transformant was incubated at 37 °C in HCHN medium for 7 days. The amount of CDH secreted was ∼50% of rCDH cultures, based on the DCPIP reduction assay. Western blot analysis of the extracellular medium over the 7-day-old culture period indicated the presence of a 90-kDa CDH-like protein. The M65H protein was purified to homogeneity by cellulose affinity chromatography, gel filtration, and anionic exchange chromatography. The R z value (A 411/A 280) was 0.77, and the extinction coefficient of the Soret maximum at 411 nm was 133 mm–1 cm–1.Steady State Kinetics—Measuring CDH activity in the extracellular medium of M65H transformants suggested that the cytochrome variant efficiently reduced DCPIP, but its ability to reduce cyt c was significantly impaired. Under steady state conditions, linear double-reciprocal plots were obtained in 20 mm sodium succinate, pH 4.5, for the purified variant and for the rCDH protein. The apparent Km values for cellobiose and DCPIP and k cat values for cellobiose oxidation and DCPIP reduction were similar for both CDH proteins (Table I). However, the specific activity for cyt c reduction by the M65H variant was ∼100-fold lower than that for rCDH (Table I).Table ISteady state parameters for rCDH and the M65H variant at pH 4.5EnzymeCellobiose oxidation (DCPIP)DCPIP reductionk catKmk cat/Kmk catKmk cat/Kms-1μmm-1 s-1s-1μmm-1 s-1rCDH25.7406.4·10527.06.44.2·106M65H26.0357.6·10525.07.43.4·106EnzymeCellobiose oxidation (cyt c)Cyt c reductionk catKmk cat/Kmk catKmk cat/Kms-1μmm-1 s-1s-1μmm-1 s-1rCDH10.5185.8·10510.80.81.4·107M65H0.1NAaNA, not applicable.NA0.1NANAa NA, not applicable. Open table in a new tab UV-visible Spectroscopy of rCDH and the M65H Variant— The electronic absorption spectra for both rCDH and the M65H variant were dominated by the heme b spectrum, with a weak absorbance near 450 nm attributed to the flavin. The ferric heme spectrum of rCDH was typical for a low spin (LS) heme iron, with a Soret maximum at 421 nm and visible bands at 530 and 570 nm (Fig. 1A and Table II). The M65H substitution altered the optical properties of the ferric heme, giving rise to a spectrum that contained a mixture of LS and high spin (HS) protoheme iron signals (Table II). Moreover, the Soret band was blue-shifted to 411 nm in the variant, and the band at 730 nm, characteristic of a methionine-iron ligation (35Moore G.R. Pettigrew G.W. Cytochrome c: Evolutionary, Structural, and Physiochemical Aspects. Springer-Verlag, Berlin1990Crossref Google Scholar), disappeared. A new weak band indicative of a HS species in the ferric state is present at 630 nm. Analysis of possible heme absorbances near 500 nm was compromised by the flavin absorbance. Therefore, the truncated cytochrome domain was obtained by limited proteolysis, and the resulting electronic absorption spectrum showed a maximum at 495 and shoulders at 530 and 560 nm (Fig. 1A). As was observed with rCDH, the ferric heme in M65H is unreactive with both cyanide and imidazole (50 mm).Table IISpectral features and heme redox midpoint potential for rCDH, the M65H variant, and selected heme proteins with histidine ligationProteinOxidizedReducedE m (mV vs. normal hydrogen electrode)rCDH421, 530, 570429, 532, 562aThe addition of 400 μm cellobiose (rCDH) or a grain of dithionite (rCDH, M65H, and cytochrome domains).+164rCDH (cyt domain)421, 530, 570429, 532, 562aThe addition of 400 μm cellobiose (rCDH) or a grain of dithionite (rCDH, M65H, and cytochrome domains).+161M65H411, 530, 560, 630428, 530, 560aThe addition of 400 μm cellobiose (rCDH) or a grain of dithionite (rCDH, M65H, and cytochrome domains).-53M65H (cyt domain)411, 495, 530, 560, 630428, 530, 560aThe addition of 400 μm cellobiose (rCDH) or a grain of dithionite (rCDH, M65H, and cytochrome domains).NDbND, not determined.Cytochrome b 5cUV-visible from Ref. 51; redox potential from Ref. 40.413, 532, 560423, 526, 556+4Cytochrome b 562dUV-visible from Ref. 52; redox potential from Ref. 39.418, 529, 558427, 531, 562+167Horseradish peroxidaseeUV-visible from Ref. 53.403, 500, 641437, 556MetMbfUV-visible from Ref. 54; redox potential from Ref. 55.410, 505, 635434, 556+61a The addition of 400 μm cellobiose (rCDH) or a grain of dithi
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