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. 1999 Oct;77(4):2153–2174. doi: 10.1016/S0006-3495(99)77056-6

Crystal structures of myoglobin-ligand complexes at near-atomic resolution.

J Vojtechovský 1, K Chu 1, J Berendzen 1, R M Sweet 1, I Schlichting 1
PMCID: PMC1300496  PMID: 10512835

Abstract

We have used x-ray crystallography to determine the structures of sperm whale myoglobin (Mb) in four different ligation states (unligated, ferric aquomet, oxygenated, and carbonmonoxygenated) to a resolution of better than 1.2 A. Data collection and analysis were performed in as much the same way as possible to reduce model bias in differences between structures. The structural differences among the ligation states are much smaller than previously estimated, with differences of <0.25 A root-mean-square deviation among all atoms. One structural parameter previously thought to vary among the ligation states, the proximal histidine (His-93) azimuthal angle, is nearly identical in all the ferrous complexes, although the tilt of the proximal histidine is different in the unligated form. There are significant differences, however, in the heme geometry, in the position of the heme in the pocket, and in the distal histidine (His-64) conformations. In the CO complex the majority conformation of ligand is at an angle of 18 +/- 3 degrees with respect to the heme plane, with a geometry similar to that seen in encumbered model compounds; this angle is significantly smaller than reported previously by crystallographic studies on monoclinic Mb crystals, but still significantly larger than observed by photoselection. The distal histidine in unligated Mb and in the dioxygenated complex is best described as having two conformations. Two similar conformations are observed in MbCO, in addition to another conformation that has been seen previously in low-pH structures where His-64 is doubly protonated. We suggest that these conformations of the distal histidine correspond to the different conformational substates of MbCO and MbO(2) seen in vibrational spectra. Full-matrix refinement provides uncertainty estimates of important structural parameters. Anisotropic refinement yields information about correlated disorder of atoms; we find that the proximal (F) helix and heme move approximately as rigid bodies, but that the distal (E) helix does not.

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Selected References

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  1. Alben J. O., Beece D., Bowne S. F., Doster W., Eisenstein L., Frauenfelder H., Good D., McDonald J. D., Marden M. C., Moh P. P. Infrared spectroscopy of photodissociated carboxymyoglobin at low temperatures. Proc Natl Acad Sci U S A. 1982 Jun;79(12):3744–3748. doi: 10.1073/pnas.79.12.3744. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Anderton C. L., Hester R. E., Moore J. N. A chemometric analysis of the resonance Raman spectra of mutant carbonmonoxy-myoglobins reveals the effects of polarity. Biochim Biophys Acta. 1997 Mar 7;1338(1):107–120. doi: 10.1016/s0167-4838(96)00194-x. [DOI] [PubMed] [Google Scholar]
  3. Ansari A., Berendzen J., Braunstein D., Cowen B. R., Frauenfelder H., Hong M. K., Iben I. E., Johnson J. B., Ormos P., Sauke T. B. Rebinding and relaxation in the myoglobin pocket. Biophys Chem. 1987 May 9;26(2-3):337–355. doi: 10.1016/0301-4622(87)80034-0. [DOI] [PubMed] [Google Scholar]
  4. Austin R. H., Beeson K. W., Eisenstein L., Frauenfelder H., Gunsalus I. C. Dynamics of ligand binding to myoglobin. Biochemistry. 1975 Dec 2;14(24):5355–5373. doi: 10.1021/bi00695a021. [DOI] [PubMed] [Google Scholar]
  5. Barlow C. H., Maxwell J. C., Wallace W. J., Caughey W. S. Elucidation of the mode of binding of oxygen to iron in oxyhemoglobin by in frared spectroscopy. Biochem Biophys Res Commun. 1973 Nov 1;55(1):91–96. doi: 10.1016/s0006-291x(73)80063-4. [DOI] [PubMed] [Google Scholar]
  6. Bernstein F. C., Koetzle T. F., Williams G. J., Meyer E. F., Jr, Brice M. D., Rodgers J. R., Kennard O., Shimanouchi T., Tasumi M. The Protein Data Bank: a computer-based archival file for macromolecular structures. J Mol Biol. 1977 May 25;112(3):535–542. doi: 10.1016/s0022-2836(77)80200-3. [DOI] [PubMed] [Google Scholar]
  7. Bianconi A., Congiu-Castellano A., Dell'Ariccia M., Giovannelli A., Burattini E., Durham P. J. Increase of the Fe effective charge in hemoproteins during oxygenation process. Biochem Biophys Res Commun. 1985 Aug 30;131(1):98–102. doi: 10.1016/0006-291x(85)91775-9. [DOI] [PubMed] [Google Scholar]
  8. Brünger A. T., Krukowski A., Erickson J. W. Slow-cooling protocols for crystallographic refinement by simulated annealing. Acta Crystallogr A. 1990 Jul 1;46(Pt 7):585–593. doi: 10.1107/s0108767390002355. [DOI] [PubMed] [Google Scholar]
  9. Brünger A. T., Kuriyan J., Karplus M. Crystallographic R factor refinement by molecular dynamics. Science. 1987 Jan 23;235(4787):458–460. doi: 10.1126/science.235.4787.458. [DOI] [PubMed] [Google Scholar]
  10. Carver T. E., Brantley R. E., Jr, Singleton E. W., Arduini R. M., Quillin M. L., Phillips G. N., Jr, Olson J. S. A novel site-directed mutant of myoglobin with an unusually high O2 affinity and low autooxidation rate. J Biol Chem. 1992 Jul 15;267(20):14443–14450. [PubMed] [Google Scholar]
  11. Chance M. R., Courtney S. H., Chavez M. D., Ondrias M. R., Friedman J. M. O2 and CO reactions with heme proteins: quantum yields and geminate recombination on picosecond time scales. Biochemistry. 1990 Jun 12;29(23):5537–5545. doi: 10.1021/bi00475a018. [DOI] [PubMed] [Google Scholar]
  12. Cheng X. D., Schoenborn B. P. Neutron diffraction study of carbonmonoxymyoglobin. J Mol Biol. 1991 Jul 20;220(2):381–399. doi: 10.1016/0022-2836(91)90020-7. [DOI] [PubMed] [Google Scholar]
  13. Christian J. F., Unno M., Sage J. T., Champion P. M., Chien E., Sligar S. G. Spectroscopic effects of polarity and hydration in the distal heme pocket of deoxymyoglobin. Biochemistry. 1997 Sep 16;36(37):11198–11204. doi: 10.1021/bi9710075. [DOI] [PubMed] [Google Scholar]
  14. Decatur S. M., Boxer S. G. A test of the role of electrostatic interactions in determining the CO stretch frequency in carbonmonoxymyoglobin. Biochem Biophys Res Commun. 1995 Jul 6;212(1):159–164. doi: 10.1006/bbrc.1995.1950. [DOI] [PubMed] [Google Scholar]
  15. Feitelson J., Yedgar S. The effect of the solvent viscosity on the migration of small molecules through the structure of myoglobin. Biorheology. 1991;28(1-2):99–105. doi: 10.3233/bir-1991-281-210. [DOI] [PubMed] [Google Scholar]
  16. Frauenfelder H., Sligar S. G., Wolynes P. G. The energy landscapes and motions of proteins. Science. 1991 Dec 13;254(5038):1598–1603. doi: 10.1126/science.1749933. [DOI] [PubMed] [Google Scholar]
  17. Frazão C., Soares C. M., Carrondo M. A., Pohl E., Dauter Z., Wilson K. S., Hervás M., Navarro J. A., De la Rosa M. A., Sheldrick G. M. Ab initio determination of the crystal structure of cytochrome c6 and comparison with plastocyanin. Structure. 1995 Nov 15;3(11):1159–1169. doi: 10.1016/s0969-2126(01)00252-0. [DOI] [PubMed] [Google Scholar]
  18. Fuchsman W. H., Appleby C. A. CO and O2 complexes of soybean leghemoglobins: pH effects upon infrared and visible spectra. Comparisons with CO and O2 complexes of myoglobin and hemoglobin. Biochemistry. 1979 Apr 3;18(7):1309–1321. doi: 10.1021/bi00574a030. [DOI] [PubMed] [Google Scholar]
  19. García A. E., Krumhansl J. A., Frauenfelder H. Variations on a theme by Debye and Waller: from simple crystals to proteins. Proteins. 1997 Oct;29(2):153–160. [PubMed] [Google Scholar]
  20. Hanson J. C., Schoenborn B. P. Real space refinement of neutron diffraction data from sperm whale carbonmonoxymyoglobin. J Mol Biol. 1981 Nov 25;153(1):117–146. doi: 10.1016/0022-2836(81)90530-1. [DOI] [PubMed] [Google Scholar]
  21. Hofrichter J., Eaton W. A. Linear dichroism of biological chromophores. Annu Rev Biophys Bioeng. 1976;5:511–560. doi: 10.1146/annurev.bb.05.060176.002455. [DOI] [PubMed] [Google Scholar]
  22. Huang X., Boxer S. G. Discovery of new ligand binding pathways in myoglobin by random mutagenesis. Nat Struct Biol. 1994 Apr;1(4):226–229. doi: 10.1038/nsb0494-226. [DOI] [PubMed] [Google Scholar]
  23. Jewsbury P., Kitagawa T. Distal residue-CO interaction in carbonmonoxy myoglobins: a molecular dynamics study of three distal mutants. Biophys J. 1995 Apr;68(4):1283–1294. doi: 10.1016/S0006-3495(95)80302-4. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Jewsbury P., Kitagawa T. The distal residue-CO interaction in carbonmonoxy myoglobins: a molecular dynamics study of two distal histidine tautomers. Biophys J. 1994 Dec;67(6):2236–2250. doi: 10.1016/S0006-3495(94)80708-8. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Jeyarajah S., Proniewicz L. M., Bronder H., Kincaid J. R. Low frequency vibrational modes of oxygenated myoglobin, hemoglobins, and modified derivatives. J Biol Chem. 1994 Dec 9;269(49):31047–31050. [PubMed] [Google Scholar]
  26. Jones T. A., Zou J. Y., Cowan S. W., Kjeldgaard M. Improved methods for building protein models in electron density maps and the location of errors in these models. Acta Crystallogr A. 1991 Mar 1;47(Pt 2):110–119. doi: 10.1107/s0108767390010224. [DOI] [PubMed] [Google Scholar]
  27. Kuriyan J., Wilz S., Karplus M., Petsko G. A. X-ray structure and refinement of carbon-monoxy (Fe II)-myoglobin at 1.5 A resolution. J Mol Biol. 1986 Nov 5;192(1):133–154. doi: 10.1016/0022-2836(86)90470-5. [DOI] [PubMed] [Google Scholar]
  28. Kushkuley B., Stavrov S. S. Theoretical study of the electrostatic and steric effects on the spectroscopic characteristics of the metal-ligand unit of heme proteins. 2. C-O vibrational frequencies, 17O isotropic chemical shifts, and nuclear quadrupole coupling constants. Biophys J. 1997 Feb;72(2 Pt 1):899–912. doi: 10.1016/s0006-3495(97)78724-1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Lang G., Marshall W. Mössbauer effect in some haemoglobin compounds. J Mol Biol. 1966 Jul;18(3):385–404. doi: 10.1016/s0022-2836(66)80032-3. [DOI] [PubMed] [Google Scholar]
  30. Li T., Quillin M. L., Phillips G. N., Jr, Olson J. S. Structural determinants of the stretching frequency of CO bound to myoglobin. Biochemistry. 1994 Feb 15;33(6):1433–1446. doi: 10.1021/bi00172a021. [DOI] [PubMed] [Google Scholar]
  31. Lim M., Jackson T. A., Anfinrud P. A. Binding of CO to myoglobin from a heme pocket docking site to form nearly linear Fe-C-O. Science. 1995 Aug 18;269(5226):962–966. doi: 10.1126/science.7638619. [DOI] [PubMed] [Google Scholar]
  32. Makinen M. W., Houtchens R. A., Caughey W. S. Structure of carboxymyoglobin in crystals and in solution. Proc Natl Acad Sci U S A. 1979 Dec;76(12):6042–6046. doi: 10.1073/pnas.76.12.6042. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Maxwell J. C., Caughey W. S. An infrared study of NO bonding to heme B and hemoglobin A. Evidence for inositol hexaphosphate induced cleavage of proximal histidine to iron bonds. Biochemistry. 1976 Jan 27;15(2):388–396. doi: 10.1021/bi00647a023. [DOI] [PubMed] [Google Scholar]
  34. Miller L. M., Chance M. R. Structural and electronic factors that influence oxygen affinities: a spectroscopic comparison of ferrous and cobaltous oxymyoglobin. Biochemistry. 1995 Aug 15;34(32):10170–10179. doi: 10.1021/bi00032a010. [DOI] [PubMed] [Google Scholar]
  35. Moore J. N., Hansen P. A., Hochstrasser R. M. Iron-carbonyl bond geometries of carboxymyoglobin and carboxyhemoglobin in solution determined by picosecond time-resolved infrared spectroscopy. Proc Natl Acad Sci U S A. 1988 Jul;85(14):5062–5066. doi: 10.1073/pnas.85.14.5062. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Mourant J. R., Braunstein D. P., Chu K., Frauenfelder H., Nienhaus G. U., Ormos P., Young R. D. Ligand binding to heme proteins: II. Transitions in the heme pocket of myoglobin. Biophys J. 1993 Oct;65(4):1496–1507. doi: 10.1016/S0006-3495(93)81218-9. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Nienhaus G. U., Chu K., Jesse K. Structural heterogeneity and ligand binding in carbonmonoxy myoglobin crystals at cryogenic temperatures. Biochemistry. 1998 May 12;37(19):6819–6823. doi: 10.1021/bi972843h. [DOI] [PubMed] [Google Scholar]
  38. Olson J. S., Phillips G. N., Jr Kinetic pathways and barriers for ligand binding to myoglobin. J Biol Chem. 1996 Jul 26;271(30):17593–17596. doi: 10.1074/jbc.271.30.17593. [DOI] [PubMed] [Google Scholar]
  39. Ormos P., Braunstein D., Frauenfelder H., Hong M. K., Lin S. L., Sauke T. B., Young R. D. Orientation of carbon monoxide and structure-function relationship in carbonmonoxymyoglobin. Proc Natl Acad Sci U S A. 1988 Nov;85(22):8492–8496. doi: 10.1073/pnas.85.22.8492. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Osapay K., Theriault Y., Wright P. E., Case D. A. Solution structure of carbonmonoxy myoglobin determined from nuclear magnetic resonance distance and chemical shift constraints. J Mol Biol. 1994 Nov 25;244(2):183–197. doi: 10.1006/jmbi.1994.1718. [DOI] [PubMed] [Google Scholar]
  41. Park K. D., Guo K. M., Adebodun F., Chiu M. L., Sligar S. G., Oldfield E. Distal and proximal ligand interactions in heme proteins: correlations between C-O and Fe-C vibrational frequencies, oxygen-17 and carbon-13 nuclear magnetic resonance chemical shifts, and oxygen-17 nuclear quadrupole coupling constants in C17O- and 13CO-labeled species. Biochemistry. 1991 Mar 5;30(9):2333–2347. doi: 10.1021/bi00223a007. [DOI] [PubMed] [Google Scholar]
  42. Pauling L., Coryell C. D. The Magnetic Properties and Structure of Hemoglobin, Oxyhemoglobin and Carbonmonoxyhemoglobin. Proc Natl Acad Sci U S A. 1936 Apr;22(4):210–216. doi: 10.1073/pnas.22.4.210. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Peng S. M., Ibers J. A. Stereochemistry of carbonylmetalloporphyrins. The structure of (pyridine)(carbonyl)(5, 10, 15, 20-tetraphenylprophinato)iron(II). J Am Chem Soc. 1976 Dec 8;98(25):8032–8036. doi: 10.1021/ja00441a025. [DOI] [PubMed] [Google Scholar]
  44. Phillips G. N., Jr, Pettitt B. M. Structure and dynamics of the water around myoglobin. Protein Sci. 1995 Feb;4(2):149–158. doi: 10.1002/pro.5560040202. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Phillips S. E., Schoenborn B. P. Neutron diffraction reveals oxygen-histidine hydrogen bond in oxymyoglobin. Nature. 1981 Jul 2;292(5818):81–82. doi: 10.1038/292081a0. [DOI] [PubMed] [Google Scholar]
  46. Phillips S. E. Structure and refinement of oxymyoglobin at 1.6 A resolution. J Mol Biol. 1980 Oct 5;142(4):531–554. doi: 10.1016/0022-2836(80)90262-4. [DOI] [PubMed] [Google Scholar]
  47. Potter W. T., Tucker M. P., Houtchens R. A., Caughey W. S. Oxygen infrared spectra of oxyhemoglobins and oxymyoglobins. Evidence of two major liganded O2 structures. Biochemistry. 1987 Jul 28;26(15):4699–4707. doi: 10.1021/bi00389a016. [DOI] [PubMed] [Google Scholar]
  48. Powers L., Sessler J. L., Woolery G. L., Chance B. CO bond angle changes in photolysis of carboxymyoglobin. Biochemistry. 1984 Nov 6;23(23):5519–5523. doi: 10.1021/bi00318a021. [DOI] [PubMed] [Google Scholar]
  49. Quillin M. L., Arduini R. M., Olson J. S., Phillips G. N., Jr High-resolution crystal structures of distal histidine mutants of sperm whale myoglobin. J Mol Biol. 1993 Nov 5;234(1):140–155. doi: 10.1006/jmbi.1993.1569. [DOI] [PubMed] [Google Scholar]
  50. Sage J. T., Jee W. Structural characterization of the myoglobin active site using infrared crystallography. J Mol Biol. 1997 Nov 21;274(1):21–26. doi: 10.1006/jmbi.1997.1367. [DOI] [PubMed] [Google Scholar]
  51. Scheidt W. R., Haller K. J., Fons M., Mashiko T., Reed C. A. A (carbonmonoxy)heme complex with a weak proximal bond. Molecular stereochemistry of carbonyl(deuteroporphinato)(tetrahydrofuran)iron(II). Biochemistry. 1981 Jun 9;20(12):3653–3657. doi: 10.1021/bi00515a054. [DOI] [PubMed] [Google Scholar]
  52. Schlichting I., Berendzen J., Phillips G. N., Jr, Sweet R. M. Crystal structure of photolysed carbonmonoxy-myoglobin. Nature. 1994 Oct 27;371(6500):808–812. doi: 10.1038/371808a0. [DOI] [PubMed] [Google Scholar]
  53. Takano T. Structure of myoglobin refined at 2-0 A resolution. I. Crystallographic refinement of metmyoglobin from sperm whale. J Mol Biol. 1977 Mar 5;110(3):537–568. doi: 10.1016/s0022-2836(77)80111-3. [DOI] [PubMed] [Google Scholar]
  54. Takano T. Structure of myoglobin refined at 2-0 A resolution. II. Structure of deoxymyoglobin from sperm whale. J Mol Biol. 1977 Mar 5;110(3):569–584. doi: 10.1016/s0022-2836(77)80112-5. [DOI] [PubMed] [Google Scholar]
  55. Terwilliger T. C., Berendzen J. Bayesian difference refinement. Acta Crystallogr D Biol Crystallogr. 1996 Sep 1;52(Pt 5):1004–1011. doi: 10.1107/S0907444996006725. [DOI] [PubMed] [Google Scholar]
  56. Terwilliger T. C., Berendzen J. Bayesian weighting for macromolecular crystallographic refinement. Acta Crystallogr D Biol Crystallogr. 1996 Jul 1;52(Pt 4):743–748. doi: 10.1107/S0907444996001473. [DOI] [PubMed] [Google Scholar]
  57. Tilton R. F., Jr, Kuntz I. D., Jr, Petsko G. A. Cavities in proteins: structure of a metmyoglobin-xenon complex solved to 1.9 A. Biochemistry. 1984 Jun 19;23(13):2849–2857. doi: 10.1021/bi00308a002. [DOI] [PubMed] [Google Scholar]
  58. Tolman J. R., Flanagan J. M., Kennedy M. A., Prestegard J. H. NMR evidence for slow collective motions in cyanometmyoglobin. Nat Struct Biol. 1997 Apr;4(4):292–297. doi: 10.1038/nsb0497-292. [DOI] [PubMed] [Google Scholar]
  59. Tsubaki M., Yu N. T. Resonance Raman investigation of dioxygen bonding in oxycobaltmyoglobin and oxycobalthemoglobin: structural implication of splittings of the bound O--O stretching vibration. Proc Natl Acad Sci U S A. 1981 Jun;78(6):3581–3585. doi: 10.1073/pnas.78.6.3581. [DOI] [PMC free article] [PubMed] [Google Scholar]
  60. WEISS J. J. NATURE OF THE IRON-OXYGEN BOND IN OXYHAEMOGLOBIN. Nature. 1964 Apr 4;202:83–84. doi: 10.1038/202083b0. [DOI] [PubMed] [Google Scholar]
  61. Weissbluth M., Maling J. E. Interpretation of quadrupole splittings and isomer shifts in hemoglobin. J Chem Phys. 1967 Nov 15;47(10):4166–4172. doi: 10.1063/1.1701594. [DOI] [PubMed] [Google Scholar]
  62. Wilbur D. J., Allerhand A. Titration behavior and tautomeric states of individual histidine residues of myoglobins. Application of natural abundance carbon 13 nuclear magnetic resonance spectroscopy. J Biol Chem. 1977 Jul 25;252(14):4968–4975. [PubMed] [Google Scholar]
  63. Yang F., Phillips G. N., Jr Crystal structures of CO-, deoxy- and met-myoglobins at various pH values. J Mol Biol. 1996 Mar 8;256(4):762–774. doi: 10.1006/jmbi.1996.0123. [DOI] [PubMed] [Google Scholar]
  64. van Aalten D. M., Conn D. A., de Groot B. L., Berendsen H. J., Findlay J. B., Amadei A. Protein dynamics derived from clusters of crystal structures. Biophys J. 1997 Dec;73(6):2891–2896. doi: 10.1016/S0006-3495(97)78317-6. [DOI] [PMC free article] [PubMed] [Google Scholar]

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