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• W2151621350 abstract "High resolution crystal structures of myoglobin in the pH range 5.2–8.7 have been used as models for the peroxide-derived compound II intermediates in heme peroxidases and oxygenases. The observed Fe–O bond length (1.86–1.90 Å) is consistent with that of a single bond. The compound II state of myoglobin in crystals was controlled by single-crystal microspectrophotometry before and after synchrotron data collection. We observe some radiation-induced changes in both compound II (resulting in intermediate H) and in the resting ferric state of myoglobin. These radiation-induced states are quite unstable, and compound II and ferric myoglobin are immediately regenerated through a short heating above the glass transition temperature (<1 s) of the crystals. It is unclear how this influences our compound II structures compared with the unaffected compound II, but some crystallographic data suggest that the influence on the Fe–O bond distance is minimal. Based on our crystallographic and spectroscopic data we suggest that for myoglobin the compound II intermediate consists of an FeIV–O species with a single bond. The presence of FeIV is indicated by a small isomer shift of δ = 0.07 mm/s from Moössbauer spectroscopy. Earlier quantum refinements (crystallographic refinement where the molecular-mechanics potential is replaced by a quantum chemical calculation) and density functional theory calculations suggest that this intermediate H species is protonated. High resolution crystal structures of myoglobin in the pH range 5.2–8.7 have been used as models for the peroxide-derived compound II intermediates in heme peroxidases and oxygenases. The observed Fe–O bond length (1.86–1.90 Å) is consistent with that of a single bond. The compound II state of myoglobin in crystals was controlled by single-crystal microspectrophotometry before and after synchrotron data collection. We observe some radiation-induced changes in both compound II (resulting in intermediate H) and in the resting ferric state of myoglobin. These radiation-induced states are quite unstable, and compound II and ferric myoglobin are immediately regenerated through a short heating above the glass transition temperature (<1 s) of the crystals. It is unclear how this influences our compound II structures compared with the unaffected compound II, but some crystallographic data suggest that the influence on the Fe–O bond distance is minimal. Based on our crystallographic and spectroscopic data we suggest that for myoglobin the compound II intermediate consists of an FeIV–O species with a single bond. The presence of FeIV is indicated by a small isomer shift of δ = 0.07 mm/s from Moössbauer spectroscopy. Earlier quantum refinements (crystallographic refinement where the molecular-mechanics potential is replaced by a quantum chemical calculation) and density functional theory calculations suggest that this intermediate H species is protonated. Myoglobin (Mb) 2The abbreviations used are: Mb, myoglobin; HRP, horseradish peroxidase; P450, cytochrome P450; CPO, chloroperoxidase; CCP, cytochrome c peroxidase; rRaman, resonance Raman; DFT, density functional theory; EXAFS, extended X-ray absorption fine structure; SNBL, Swiss-Norwegian Beam Line; ESRF, European Synchrotron Radiation Facility in Grenoble, France; oxyMb, oxymyoglobin; deoxyMb, deoxymyoglobin; MOPS, 4-morpholinepropanesulfonic acid; TAPS, 3-{[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]amino}-1-propanesulfonic acid. 2The abbreviations used are: Mb, myoglobin; HRP, horseradish peroxidase; P450, cytochrome P450; CPO, chloroperoxidase; CCP, cytochrome c peroxidase; rRaman, resonance Raman; DFT, density functional theory; EXAFS, extended X-ray absorption fine structure; SNBL, Swiss-Norwegian Beam Line; ESRF, European Synchrotron Radiation Facility in Grenoble, France; oxyMb, oxymyoglobin; deoxyMb, deoxymyoglobin; MOPS, 4-morpholinepropanesulfonic acid; TAPS, 3-{[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]amino}-1-propanesulfonic acid. is a heme protein found mainly in heart and skeletal muscle (1Millikan G.A. Proc. R. Soc. Lond. B. 1937; 123: 218-241Crossref Google Scholar, 2Millikan G.A. Physiol. Rev. 1939; 19: 503-523Crossref Google Scholar, 3Wittenberg B.A. Wittenberg J.B. Annu. Rev. Physiol. 1989; 51: 857-878Crossref PubMed Google Scholar). Its function in oxygen storage and transportation has been well known for a long time, but more recently an understanding of Mb as a multifunction protein (functions as protection against oxidative damage and NO scavenging) has evolved (4Frauenfelder H. McMahon B.H. Austin R.H. Chu K. Grooves J.T. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 2370-2374Crossref PubMed Scopus (298) Google Scholar, 5Garry D.J. Kanatous S.B. Mammen P.P.A. Trends Cardiovasc. Med. 2003; 13: 111-116Crossref PubMed Scopus (52) Google Scholar, 6Brunori M. Trends Biochem. Sci. 2001; 26: 209-210Abstract Full Text Full Text PDF PubMed Scopus (193) Google Scholar, 7Alayash A.I. Patel R.P. Cashon R.E. Antioxid. Redox. Signal. 2001; 3: 313-327Crossref PubMed Google Scholar). The fact that Mb can react with hydrogen peroxide and give a ferryl species in a peroxidase-style manner (but with important differences, as will be detailed below) was studied already in the 1950s (8Keilin D. Hartree E.F. Nature. 1950; 166: 513-514Crossref PubMed Scopus (54) Google Scholar, 9George P. Irvine D.H. Nature. 1951; 168: 164-165Crossref PubMed Scopus (36) Google Scholar, 10George P. Irvine D.H. Biochem. J. 1952; 52: 511-517Crossref PubMed Scopus (197) Google Scholar). For a classic peroxidase, like horseradish peroxidase (HRP), the reaction cycle involves a two-electron oxidation-reduction (Scheme 1). In the first classic HRP step the resting ferric (FeIII) high spin (S = 5/2) form is oxidized by hydrogen peroxide to a water molecule and a heme state that is two oxidation equivalents higher (compound I) than the resting ferric form. This first step propagates through a hydroperoxo-intermediate (compound 0) where compound I is generated through a heterolytic cleavage of the O–O bond (11Ibrahim M. Denisov I.G. Makris T.M. Kincaid J.R. Sligar S.G. J. Am. Chem. Soc. 2003; 125: 13714-13718Crossref PubMed Scopus (51) Google Scholar, 12Denisov I.G. Makris T.M. Sligar S.G. J. Biol. Chem. 2002; 277: 42706-42710Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar, 13Ibrahim M. Kincaid J.R. J. Porphyrins Phthalocyanines. 2004; 8: 215-225Crossref Google Scholar). The distal His is assumed to function as an acid/base catalyst to facilitate heterolytic cleavage by accepting a proton from the inner oxygen (oxygen ligated to the iron) and then donating it to the outer (leaving) oxygen. The negative charge on the leaving hydroxide during bond cleavage is then stabilized by the distal Arg in HRP (not found in Mb) (14Poulos T.L. Kraut J. J. Biol. Chem. 1980; 255: 8199-8205Abstract Full Text PDF PubMed Google Scholar, 15Sono M. Roach M.P. Coulter E.D. Dawson J.H. Chem. Rev. 1996; 96: 2841-2887Crossref PubMed Google Scholar). Additionally, the proximal His can stabilize higher oxidation states on the heme iron by hydrogen bonding to a neighboring carboxylate group, thus making the Fe-bound nitrogen more negative (14Poulos T.L. Kraut J. J. Biol. Chem. 1980; 255: 8199-8205Abstract Full Text PDF PubMed Google Scholar, 15Sono M. Roach M.P. Coulter E.D. Dawson J.H. Chem. Rev. 1996; 96: 2841-2887Crossref PubMed Google Scholar). The compound I intermediate formed is two oxidation equivalents higher than the resting form (FeIII). One electron is withdrawn from the iron, resulting in a ferryl (FeIV = O) state with a double-bonded oxygen atom and intermediate spin state (S = 1), which could interact magnetically with radicals. Depending on the peroxidase, the second oxidizing electron comes from either the porphyrin ring, giving a π-cation radical, or from an amino acid residue (Trp or Tyr) near the heme ring (16Poulos T.L. Phil. Trans. Soc. A. 2005; 363: 793-806Crossref PubMed Scopus (32) Google Scholar, 17Everse J. Free Rad. Biol. Med. 1998; 24: 1338-1346Crossref PubMed Scopus (0) Google Scholar). In the second step of the peroxidase reaction cycle compound I carries out a substrate (e.g. organic molecules A) oxidation (A → A+·) resulting in a one-electron reduction to compound II (S = 1) by the loss of the heme/protein radical. The last step of the cycle is a further one-electron reduction of compound II to the resting ferric form accompanied again by a second substrate oxidation (A → A+·). Compound II was until a few years ago thought of as an Fe = O double bond state, but this changed when the crystal structures indicating a single-bond character were published (18Hersleth H-P. Dalhus B. Goörbitz C.H. Andersson K.K. J. Inorg. Biochem. 2001; 86 (260): 260Google Scholar, 19Berglund G.I. Carlsson G.H. Smith A.T. Szoöke H. Henriksen A. Hajdu J. Nature. 2002; 417: 463-468Crossref PubMed Scopus (642) Google Scholar). The crystal structures of compound II all indicate this single-bond character, but some recent extended x-ray absorption fine structure (EXAFS) studies lean toward a double-bond character, so the question is still under some debate (20Poulos T.L. Drug Metab. Dispos. 2005; 33: 10-18Crossref PubMed Scopus (46) Google Scholar, 21Green M.T. Dawson J.H. Gray H.B. Science. 2004; 304: 1653-1656Crossref PubMed Scopus (402) Google Scholar, 22Hersleth H-P. Ryde U. Rydberg P. Goörbitz C.H. Andersson K.K. J. Inorg. Biochem. 2006; 100: 460-476Crossref PubMed Scopus (0) Google Scholar). Mb (Scheme 1 and Fig. 1) can react with hydrogen peroxide in a manner similar to peroxidases, including oxidizing several classes of small organic molecules, but at a much lower rate (23King N.K. Winfield M.E. J. Biol. Chem. 1963; 238: 1520-1528Abstract Full Text PDF PubMed Google Scholar, 24Guilivi C. Cadenas E. Methods Enzymol. 1994; 233: 189-202Crossref PubMed Scopus (72) Google Scholar). The reduced efficiency can be explained by the lack of three essential residues; the Asp that forms the hydrogen bond to the proximal His, the distal Arg that stabilize the negative charge on the leaving group, and the distal His that is suggested to be too close to the heme to fully function as an acid-base catalyst during the heterolytic cleavage of the peroxide bond (14Poulos T.L. Kraut J. J. Biol. Chem. 1980; 255: 8199-8205Abstract Full Text PDF PubMed Google Scholar, 15Sono M. Roach M.P. Coulter E.D. Dawson J.H. Chem. Rev. 1996; 96: 2841-2887Crossref PubMed Google Scholar, 25Matsui T. Ozaki S-i. Liong E. Phillips G.N. Watanabe Jr., Y. J. Biol. Chem. 1999; 274: 2838-2844Abstract Full Text Full Text PDF PubMed Scopus (171) Google Scholar, 26Watanabe Y. Ueno T. Bull. Chem. Soc. Jpn. 2003; 76: 1309-1322Crossref Scopus (29) Google Scholar). Through mutation studies Watanabe and co-workers (25Matsui T. Ozaki S-i. Liong E. Phillips G.N. Watanabe Jr., Y. J. Biol. Chem. 1999; 274: 2838-2844Abstract Full Text Full Text PDF PubMed Scopus (171) Google Scholar, 27Ozaki S-I. Matsui T. Watanabe Y. J. Am. Chem. Soc. 1997; 119: 6666-6667Crossref Scopus (85) Google Scholar, 28Ozaki S-i. Matsui T. Roach M.P. Watanabe Y. Coord. Chem. Rev. 2000; 198: 39-59Crossref Scopus (75) Google Scholar) showed that mimicking the His distance in peroxidases increased the peroxidase activity of Mb and also led to the observation of compound I. Furthermore, a homolytic cleavage of the peroxide bond in compound 0 that gives Mb compound II and a hydroxyl radical have been suggested (29Allentoff A.J. Bolton J.L. Wilks A. Thompson J.A. Ortiz de Montellano P.R. J. Am. Chem. Soc. 1992; 114: 9744-9749Crossref Scopus (70) Google Scholar, 30Van der Zee J. Biochem. J. 1997; 322: 633-639Crossref PubMed Scopus (27) Google Scholar). The Mb compound I has been observed in deuterated water (31Egawa T. Shimada H. Ishimura Y. J. Biol. Chem. 2000; 275: 34858-34866Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar). Formation of radicals was also observed in the reaction of Mb with hydrogen peroxide (32Gibson J.F. Ingram D.J.E. Nature. 1956; 178: 871-872Crossref Scopus (31) Google Scholar). For horse Mb the semi-stable radicals were shown to be a peroxyl-radical on Trp-14 and a phenoxyl-radical at Tyr-103 (33Gunther M.R. Free Rad. Biol. Med. 2004; 36: 1345-1354Crossref PubMed Scopus (31) Google Scholar).FIGURE 1Crystal structure of the heme region in Mb compound II at pH 5.2. Structure is shown with the final electron density map 2Fo - Fc (contoured at 1σ in gold), the final electron density difference map Fo - Fc (contoured at +3σ in green and at -3σ in red), the electron density difference Fo - Fc maps with the iron-ligated oxygen atom omitted for map calculation (contoured at 5σ in magenta), and the electron density difference map Fo - Fc (contoured at +3σ in cyan) before adding the glycerol.View Large Image Figure ViewerDownload Hi-res image Download (PPT) The peroxidase intermediates described above are also found in heme-based oxygenases and catalases even though their reaction cycles are different. Several crystal structures of these intermediates have been solved (22Hersleth H-P. Ryde U. Rydberg P. Goörbitz C.H. Andersson K.K. J. Inorg. Biochem. 2006; 100: 460-476Crossref PubMed Scopus (0) Google Scholar). As mentioned above, active site amino acids of peroxidases are involved in deprotonation/protonation of the iron-linked peroxide in the first step of the reaction cycle. In contrast, it appears that for the monooxygenases cytochrome P450 (P450), nitric-oxide synthase, and heme oxygenases it is the solvent molecules that are responsible for the protonation of the dioxygen leading to the cleavage of the O–O bond (34Poulos T.L. Biochem. Biophys. Res. Commun. 2005; 338: 337-345Crossref PubMed Scopus (64) Google Scholar). For P450 and nitric-oxide synthase as well as for bromo- and chloroperoxidase (CPO) the proximal ligand is not a His, but a Cys, and for most catalases it is a Tyr. These three residues differ in their electron donating capability to the iron, which can lead to some differences between these enzymes. There have been some conflicting reports through the years regarding the two pH-dependent structural forms of compound II (35Andersson L.A. Dawson J.H. Struct. Bonding. 1990; 64: 1-40Google Scholar). For HRP two different distances for the Fe–O bond length in compound II (1.6 and 1.9 Å, respectively) were observed in EXAFS studies and suggested to be due the pH dependence of compound II (35Andersson L.A. Dawson J.H. Struct. Bonding. 1990; 64: 1-40Google Scholar, 36Chance B. Powers L. Ching Y. Poulos T.L. Schonbaum G.R. Yamazaki I. Paul K.G. Arch. Biochem. Biophys. 1984; 235: 596-611Crossref PubMed Google Scholar, 37Penner-Hahn J.E. Eble K.S. McMurry T.J. Renner M. Balch A.L. Groves J.T. Dawson J.H. Hodgson K.O. J. Am. Chem. Soc. 1986; 108: 7819-7825Crossref PubMed Google Scholar, 38Chang C.S. Yamazaki I. Sinclair R. Khalid S. Powers L. Biochemistry. 1993; 32: 923-928Crossref PubMed Scopus (37) Google Scholar). For Mb different spectroscopic products have been observed at high and low pH in the reaction with hydrogen peroxide (39King N.K. Winfield M.E. Aust. J. Biol. Sci. 1966; 19: 211-217Crossref PubMed Google Scholar, 40Foote N. Gadsby P.M.A. Greenwood C. Thomson A.J. Biochem. J. 1989; 261: 515-522Crossref PubMed Scopus (26) Google Scholar, 41Wittenberg J.B. J. Biol. Chem. 1978; 253: 5694-5695Abstract Full Text PDF PubMed Google Scholar). The ferryl state of Mb is important, because it is believed to be of physiological relevance through its involvement in oxidative stress reactions (5Garry D.J. Kanatous S.B. Mammen P.P.A. Trends Cardiovasc. Med. 2003; 13: 111-116Crossref PubMed Scopus (52) Google Scholar, 7Alayash A.I. Patel R.P. Cashon R.E. Antioxid. Redox. Signal. 2001; 3: 313-327Crossref PubMed Google Scholar, 42Reeder B.J. Svistunenko D.A. Cooper C.E. Wilson M.T. Antioxid. Redox. Signal. 2004; 6: 954-966Crossref PubMed Scopus (133) Google Scholar). During ischemia oxymyoglobin (oxyMb) is converted to ferrous deoxymyoglobin (deoxyMb), which in turn is much more easily oxidized during reperfusion when reactive oxygen species are formed, initially to ferric Mb and then further to ferryl Mb (43Arduini A. Hochstein P. Oxidative Damage & Repair. 1991; (Davies, K. J., ed) pp. , Pergamon, Oxford: 406-414Google Scholar, 44Galaris D. Eddy L. Arduini A. Cadenas E. Hochstein P. Biochem. Biophys. Res. Commun. 1989; 160: 1162-1168Crossref PubMed Google Scholar). This ferryl Mb can oxidize essential biological constituents (44Galaris D. Eddy L. Arduini A. Cadenas E. Hochstein P. Biochem. Biophys. Res. Commun. 1989; 160: 1162-1168Crossref PubMed Google Scholar), but it can be reduced back to ferric Mb if the supply of reductants is sufficient (43Arduini A. Hochstein P. Oxidative Damage & Repair. 1991; (Davies, K. J., ed) pp. , Pergamon, Oxford: 406-414Google Scholar) and thereby plays a defensive role in protection of the heart by removal of reactive oxygen species (45Floögel U. Goödecke A. Klotz L-O. Schrader J. FASEB J. 2004; 18: 1156-1158Crossref PubMed Google Scholar). It has also been suggested that Mb can function as a detoxifier of both H2O2 and NO (46Herold S. Rehmann F-J.K. J. Biol. Inorg. Chem. 2001; 6: 543-555Crossref PubMed Scopus (76) Google Scholar). High valent Mb may, however, have harmful effects by oxidizing lipids and be involved in muscle injury and subsequent renal failure, in which heme to protein cross-linked Mb and acidic pH play an important role (42Reeder B.J. Svistunenko D.A. Cooper C.E. Wilson M.T. Antioxid. Redox. Signal. 2004; 6: 954-966Crossref PubMed Scopus (133) Google Scholar, 47Moore K.P. Holt S.G. Patel R.P. Svistunenko D.A. Zackert W. Goodier D. Reeder B.J. Clozel M. Anand R. Cooper C.E. Morrow J.D. Wilson M.T. Darley-Usmar V. Roberts II L.J. J. Biol. Chem. 1998; 273: 31731-31737Abstract Full Text Full Text PDF PubMed Scopus (229) Google Scholar). In the present study we try to elucidate the structure of Mb compound II by the use of crystallography in combination with spectroscopy and previous theoretical studies (22Hersleth H-P. Ryde U. Rydberg P. Goörbitz C.H. Andersson K.K. J. Inorg. Biochem. 2006; 100: 460-476Crossref PubMed Scopus (0) Google Scholar, 48Nilsson K. Hersleth H-P. Rod T.H Andersson K.K Ryde U. Biophys. J. 2004; 87: 3437-3447Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar). In the crystallographic studies the potential radiation damage and radiation-induced reduction of the metal-center must be addressed. The use of light absorption on small protein crystals, so-called single-crystal microspectrophotometry, is essential for generation of the correct oxidation state, and for monitoring changes of this state during data collection for proteins (19Berglund G.I. Carlsson G.H. Smith A.T. Szoöke H. Henriksen A. Hajdu J. Nature. 2002; 417: 463-468Crossref PubMed Scopus (642) Google Scholar, 49Srajer V. Ren Z. Teng T-Y. Schmidt M. Ursby T. Bourgeois D. Pradervand C. Schildkamp W. Wulff M. Moffat K. Biochemistry. 2001; 40: 13802-13815Crossref PubMed Scopus (278) Google Scholar, 50Wilmot C.M. Sjoögren T. Carlsson G.H. Berglund G.I. Hajdu J. Methods Enzymol. 2002; 353: 301-318Crossref PubMed Scopus (36) Google Scholar). About 90% of the interacting x-ray photons deposit their energy into the crystal lattice mainly through the photoelectric effect by generating electrons (50Wilmot C.M. Sjoögren T. Carlsson G.H. Berglund G.I. Hajdu J. Methods Enzymol. 2002; 353: 301-318Crossref PubMed Scopus (36) Google Scholar). This, together with further secondary electron emission, produces reactive species that can lead to radiation damage. The x-ray diffraction experiment then generates potential reducing equivalents (electrons) that can change the redox-state of metal-proteins (51Carugo O. Carugo K.D. Trends Biochem. Sci. 2005; 30: 213-219Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar). The importance of these questions and the use of single-crystal microspectrophotometry was acknowledged early in the study of these peroxidase, oxygenase, and catalase intermediates (52Jouve H-M. Andreoletti P. Gouet P. Hajdu J. Gagnon J. Biochimie (Paris). 1997; 79: 667-671Crossref PubMed Scopus (20) Google Scholar, 53Fuöloöp V. Phizackerley R.P. Soltis S.M. Clifton I.J. Watatsuki S. Erman J. Hajdu J. Edwards S.L. Structure. 1994; 2: 201-208Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar, 54Gouet P. Jouve H-M. Williams P.A Andersson I. Andreoletti P. Nussaume L. Hajdu J. Nat. Struct. Biol. 1996; 3: 951-956Crossref PubMed Scopus (76) Google Scholar). The actual rate by which these intermediates are reduced varies considerably among proteins (22Hersleth H-P. Ryde U. Rydberg P. Goörbitz C.H. Andersson K.K. J. Inorg. Biochem. 2006; 100: 460-476Crossref PubMed Scopus (0) Google Scholar). The use of single-crystal microspectrophotometry is therefore essential to determine the redox-state correctly in each case. The use of crystallography in combination with microspectrophotometry, resonance Raman (rRaman), and Moössbauer spectroscopy suggests that compound II has a single FeIV–O bond and is probably protonated. Purification and Crystallization—Horse heart Mb (Sigma) was further purified by gel filtration on a Sephadex G75 column using the absorption ratio of 410/280 nm as the purification criterion. Mb was crystallized at three pH values: 5.2, 6.8, and 8.7. The crystals were grown at room temperature by batch methods with a 6–12 mg/ml Mb concentration and 80–85% of the crystallization stock solution (55Hersleth H-P. Dalhus B. Goörbitz C.H. Andersson K.K. J. Biol. Inorg. Chem. 2002; 7: 299-304Crossref PubMed Scopus (50) Google Scholar, 56Sherwood C. Mauk A.G. Brayer G.D. J. Mol. Biol. 1987; 193: 227Crossref PubMed Scopus (4) Google Scholar). The stock solution contained 3.9 m ammonium sulfate, 0.1 m buffer depending on the pH and 5–10% of glycerol. No buffer was applied at pH 5.2, whereas MOPS or HEPES was used at pH 6.8 and TAPS at pH 8.7. Rosette-shaped clusters of thin, plate-shaped crystals were grown within 1–7 days. X-ray Data Collection—For each x-ray diffraction experiment a single crystal was separated from a rosette and transferred into a cryo-solution containing 75–80% crystallization stock solution and 20–25% glycerol. Compound II intermediates were prepared by 30 s of incubation in a cryo-solution to which had been added H2O2 to a final concentration of 22 mm, with subsequent flash freezing in liquid nitrogen. The diffraction data were mainly collected at the Swiss-Norwegian Beam Line (SNBL) BM01A at the European Synchrotron Radiation Facility (ESRF), Grenoble, France, but also at beamline ID14-3 at ESRF and at beamline X11 at EMBL, DESY, Hamburg, Germany (Table 1). Usually, two separate scans, at high and low resolution, were collected using at SNBL a MAR345 image plate, at ID14-3 a ADSC Q4R charge-coupled device detector and at X11 a MARCCD 165-mm detector. Data collection temperatures were in the range of 100–110 K.TABLE 1Crystal data, data collection, and refinement statisticsRadiation-induced Mb comp. IIRadiation-induced ferric MbRadiation-induced Mb comp. IIFerrous deoxy MbRadiation-induced ferric MbRadiation-induced Mb comp IIRadiation-induced ferric MbpH5.25.26.86.86.88.78.7Crystal dataSpace groupP21P21P21P21P21P21P21a (Å)62.963.263.162.962.963.062.7b (Å)28.728.728.728.828.828.728.5c (Å)35.435.635.535.435.335.635.5β (°)105.9105.8105.9105.7106.1105.7106.2Data collectionX-ray sourceESRF-BM01AESRF-BM01AESRF-BM01AESRF-BM01AESRF-ID14-3ESRF-BM01AEMBL-X11Wavelength (Å)0.87300.87270.80000.90000.93120.87270.8496Temperature (K)100110100110100110100Resolution range (Å)20.8-1.3530.4-1.3026.8-1.3030.0-1.2522-1.2034.3-1.2021.9-1.40Completeness (%)aThe value before the backslash is for all data, and the value after the backslash is for the data in the highest resolution shell98.4/85.498.2/98.897.9/98.097.3/95.894.0/86.699.1/99.195.2/95.2Redundancy (%)aThe value before the backslash is for all data, and the value after the backslash is for the data in the highest resolution shell4.2/2.42.5/2.32.6/2.22.6/2.32.7/2.02.5/2.33.0/2.9I/σ(I)aThe value before the backslash is for all data, and the value after the backslash is for the data in the highest resolution shell22.0/2.713.2/4.413.5/2.516.9/4.116.4/3.412.7/2.624.2/2.8RsymbRsym = Σ|I - 〈I 〉|/ΣI8.3/30.54.2/18.34.6/31.03.5/18.04.6/34.04.4/35.54.4/28.8Refinement statisticsRcryst (%)cRcryst = Σ (|Fobs| - |Fcalc|)/Σ|Fobs|13.913.114.813.413.713.412.9Rfree (%)dRfree is the Rcryst value calculated on the 5% reflections excluded for refinement17.416.517.616.416.816.316.6Mean protein/solvent isotropic B-factor (Å2)11.3/22.413.4/31.612.4/30.114.1/30.710.6/27.011.0/29.213.3/28.7Ramachandran plot: ration in most favored/other allowed regions (%)91.8/8.292.5/7.591.0/9.091.8/8.291.8/8.291.8/8.291.8/8.2Estimated overall coordinate error based on Rcryst/maximum likelihood (Å)0.073/0.0390.056/0.0300.064/0.0340.050/0.0290.047/0.0260.044/0.0290.077/0.038Added waters190183182193218214201Volume not occupied by model (%)3.93.73.73.72.12.53.5PDB code2V1G2V1H2V1E2V1K2V1I2V1F2V1Ja The value before the backslash is for all data, and the value after the backslash is for the data in the highest resolution shellb Rsym = Σ|I - 〈I 〉|/ΣIc Rcryst = Σ (|Fobs| - |Fcalc|)/Σ|Fobs|d Rfree is the Rcryst value calculated on the 5% reflections excluded for refinement Open table in a new tab The diffraction data were processed with MOSFLM (57Alben J.O. Fiamingo F.G. Croteau A.A. Hemann C.F. Powell K.A. Molleran V.M. Park S. Physica B. 1989; 158: 87-89Crossref Scopus (2) Google Scholar) and scaled with SCALA (58Number Collaborative Computational Project Acta Crystallogr. Sect. D Biol. Crystallogr. 1994; 50: 760-763Crossref PubMed Scopus (17688) Google Scholar, 59Evans P. Sawyer L. Isaacs N. Bailey S. Proceedings of the CCP4 Study Weekend. Data Collection and Processing. 1993: 114-122Google Scholar) or processed with DENZO (60Otwinowski Z. Minor W. Methods Enzymol. 1997; 276: 307-326Crossref PubMed Scopus (36256) Google Scholar) and scaled with SCALEPACK (60Otwinowski Z. Minor W. Methods Enzymol. 1997; 276: 307-326Crossref PubMed Scopus (36256) Google Scholar) (Table 1). The first structure (PDB entry 1GJN (55Hersleth H-P. Dalhus B. Goörbitz C.H. Andersson K.K. J. Biol. Inorg. Chem. 2002; 7: 299-304Crossref PubMed Scopus (50) Google Scholar)) was solved by molecular replacement with CNS (61Bruönger 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 (16743) Google Scholar) using PDB entry 1WLA as the staring model (62Maurus R. Overall C.M. Bogumil R. Luo Y. Mauk A.G. Smith M. Brayer G.D. Biochim. Biophys. Acta. 1997; 1997: 1-13Crossref Scopus (92) Google Scholar), whereas the 1GJN structure itself served as the starting model for the other structures (55Hersleth H-P. Dalhus B. Goörbitz C.H. Andersson K.K. J. Biol. Inorg. Chem. 2002; 7: 299-304Crossref PubMed Scopus (50) Google Scholar). Refinements, including rigid body, simulated annealing, and conjugate gradient minimizations with CNS, and model improvements in O (63Jones P. J. Biol. Chem. 2001; 276: 13791-13796Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar), were performed initially for some structures. Further steps included multiple cycles of restrained refinement in Refmac (58Number Collaborative Computational Project Acta Crystallogr. Sect. D Biol. Crystallogr. 1994; 50: 760-763Crossref PubMed Scopus (17688) Google Scholar, 64Murshudov G.N. Grebenko A.I. Brannigan J.A. Antson A.A. Barynin V.V. Dodson G.G. Dauter Z. Wilson K.S. Melik-Adamyan W.R. Acta Crystallogr. Sect. D Biol. Crystallogr. 2002; 58: 1972-1982Crossref PubMed Scopus (0) Google Scholar) and model building in Coot (65Emsley P. Cowtan K. Acta Crystallogr. Sect. D Biol. Crystallogr. 2004; 60: 2126-2132Crossref PubMed Scopus (20944) Google Scholar) and addition of water molecules by ARP/wARP (66Lamzin V.S. Wilson K.S. Acta Crystallogr. Sect. D Biol. Crystallogr. 1993; 49: 129-147Crossref PubMed Google Scholar). In the later stages TLS refinement was introduced, and finally restrained anisotropic refinement performed with Refmac (67Murshudov G.N. Lebedev A. Vagin A.A. Wilson K.S. Dodson E.J. Acta Crystallogr. Sect. D Biol. Crystallogr. 1999; 55: 249-255Crossref Scopus (990) Google Scholar, 68Winn M. Isupov M. Murshudov G.N. Acta Crystallogr. Sect. D Biol. Crystallogr. 2001; 57: 122-133Crossref PubMed Scopus (1610) Google Scholar). The anisotropy was monitored through the PARVATI program (69Merritt E.A. Acta Crystallogr. Sect. D Biol. Crystallogr. 1999; 55: 1109-1117Crossref PubMed Scopus (162) Google Scholar). Both steps lead to a significant drop in the Rcryst and Rfree values. All structures were finally refined with Refmac. No restraints were used for the Fe–NHEME, Fe–NHIS, and Fe–O distances. The same hkl reflections were selected as Rfree set for all the structures. The figures were prepared with PyMOL. 3W. L. DeLano (2002) PyMOL, DeLano Scientific, San Carlos, CA. The atomic coordinates and structure factors have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ. Microspectrophotometry—The crystals described above were transferred between the microspectrophotometer and the diffractometer with cryo tongs (Hampton Research). Measurements were carried out on a microspectrophotometer system (4DX Systems AB, Uppsala, Sweden) on SNBL, whereas earlier measurements applied the Cryo-bench in connection with ID09 at ESRF" @default.
• W2151621350 created "2016-06-24" @default.
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• W2151621350 date "2007-08-01" @default.
• W2151621350 modified "2023-10-08" @default.
• W2151621350 title "Crystallographic and Spectroscopic Studies of Peroxide-derived Myoglobin Compound II and Occurrence of Protonated FeIV–O" @default.
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