Structure of the semiquinone form of flavodoxin from Clostridum MP. Extension of 1.8 A resolution and some comparisons with the oxidized state.

As part of a series of comparisons of the structures of the three oxidation states of flavodoxin from Clostridium MP, phases for the semiquinonc form were determined to 2.0 A resolution by isomorphous replacement ( = 0.725). Subsequently, the structure was refined at 1.8 A resolution by a combination of difference Fourier, real space and reciprocal space methods. After refining to an R of 0.194, we explored the conformation of the FMN binding site by real space refinement verau6 maps with Fourier coefficients of the form (2) F,I - 1 P,I ) exp (in,). To minimize bias in the fitting, groups of atoms were systematically omitted from the structure factors used in computat.ion of the (212”,1 - IF,\) maps. One-electron reduction of oxidized flavodoxin is accompanied by several changes at the FMN binding site: the conformation of residues in the reverse bend formed by Met56-Gly57-Asp58-Glu59 differs in the crystal structures of the oxidized and semiquinone species; further, backbone atoms in residues 55 and 89 shift by more than 0.5 A and the indole ring of Trp90 undergoes a significant displacement. The orientation of the peptide unit connecting Gly57 and Asp58 is consistent with the presence of a hydrogen bond between the carbonyl oxygen of Gly57 and the flavin N(5) in flavodoxin semiquinone. No equivalent bond is found in oxidized flavodoxin. In both the oxidized and semiquinone species of clostridial flavodoxin, the isoalloxazine ring is essentially planar : the bending angles about N(5)-N( 10) are -2.5” for the semiquinone structure and ~0’ in oxidized flavodoxin. The intensity changes resulting from t,he oxidized- ,semiquinone conversion (K, m= 0.33) arise in part from changes in molecular packing. Intermolecular contacts, including neighbors of the prosthetic group, are altered in the repacking. Maps or models of the two oxidation states can be brought into approximate coincidence by

[1]  J. Le Gall,et al.  Chemical study of two flavodoxins extracted from sulfate reducing bacteria. , 1970, Biochemical and biophysical research communications.

[2]  J. Hofrichter,et al.  Optical spectra and electronic structure of flavine mononucleotide in flavodoxin crystals. , 1975, Biochemistry.

[3]  M. Haniu,et al.  The amino acid sequence of the Clostridium MP flavodoxin. , 1974, The Journal of biological chemistry.

[4]  L. Mazzarella,et al.  Structure and function of haemoglobin: IV. A three-dimensional Fourier synthesis of horse deoxyhaemoglobin at 5.5 Å resolution , 1967 .

[5]  G. Tollin,et al.  Flavin-protein interactions and the redox properties of the Shethna flavoprotein. , 1971, Biochemistry.

[6]  L. T. Eyck,et al.  Crystallographic fast Fourier transforms , 1973 .

[7]  N. Allewell,et al.  Design of a diffractometer and flow cell system for X-ray analysis of crystalline proteins with applications to the crystal chemistry of ribonuclease-S. , 1967, Journal of molecular biology.

[8]  O. Kennard,et al.  The structure of `high-energy' phosphate compounds. II. An X-ray analysis of the cyclohexylammonium phosphoenolpyruvate , 1973 .

[9]  R. Norrestam,et al.  Studies on flavin derivatives. The crystal and molecular structure of 4a-allyl-3,5,7,8,10-pentamethyl-4a,5-dihydroisoalloxazine , 1972 .

[10]  M G Rossmann,et al.  Comparison of super-secondary structures in proteins. , 1973, Journal of molecular biology.

[11]  V. Massey,et al.  On the existence of spectrally distinct classes of flavoprotein semiquinones. A new method for the quantitative production of flavoprotein semiquinones. , 1966, Biochemistry.

[12]  G. Karreman.  Contributions to quantum biology. I. Mobile electronic characteristics of riboflavin radicals , 1961 .

[13]  A. W. Hanson,et al.  The three-dimensional structure of ribonuclease-S. Interpretation of an electron density map at a nominal resolution of 2 A. , 1970, The Journal of biological chemistry.

[14]  Joyce M. Cox,et al.  Mathematical methods used in the comparison of the quaternary structures , 1967 .

[15]  V. Massey,et al.  Oxidation-reduction properties of flavodoxin from Peptostreptococcus elsdenii. , 1969, The Journal of biological chemistry.

[16]  T. Moore,et al.  Molecular luminescence studies of flavins. I. The excited states of flavins. , 1972, Journal of the American Chemical Society.

[17]  K. D. Watenpaugh,et al.  Refinement of the model of a protein: rubredoxin at 1.5 Å resolution , 1973 .

[18]  A. Ehrenberg,et al.  On the powder ESR and ENDOR spectra of flavoprotein radicals. , 1973, Biochimica et biophysica acta.

[19]  S. Mayhew.  Properties of two clostridial flavodoxins. , 1971, Biochimica et biophysica acta.

[20]  P. Song.  ELECTRONIC STRUCTURES AND SPECTRA OF FLAVINS: AN IMPROVED PARISER‐PARR‐POPLE MO AND SEMIEMPIRICAL UNRESTRICTED HARTREE‐FOCK COMPUTATIONS* , 1969, Annals of the New York Academy of Sciences.

[21]  J Deisenhofer,et al.  Structure of the complex formed by bovine trypsin and bovine pancreatic trypsin inhibitor. II. Crystallographic refinement at 1.9 A resolution. , 1974, Journal of molecular biology.

[22]  T. Ashida,et al.  The crystal structure of riboflavin hydrobromide monohydrate. , 1967, Bulletin of the Chemical Society of Japan.

[23]  H. Beinert.  Spectral Characteristics of Flavins at the Semiquinoid Oxidation Level1 , 1956 .

[24]  R. Diamond,et al.  A mathematical model-building procedure for proteins , 1966 .

[25]  P Argos,et al.  A comparison of the heme binding pocket in globins and cytochrome b5. , 1975, The Journal of biological chemistry.

[26]  M. Levitt,et al.  Energy refinement of hen egg-white lysozyme. , 1974, Journal of molecular biology.

[27]  F. S. Mathews,et al.  A semi-empirical method of absorption correction , 1968 .

[28]  R. Diamond.  A real-space refinement procedure for proteins , 1971 .

[29]  M. Sundaralingam,et al.  Stereochemistry of nucleic acid constituents: II. A comparative study , 1965 .

[30]  R. Henderson.  Structure of crystalline alpha-chymotrypsin. IV. The structure of indoleacryloyl-alpha-chyotrypsin and its relevance to the hydrolytic mechanism of the enzyme. , 1970, Journal of molecular biology.

[31]  F M Richards,et al.  The matching of physical models to three-dimensional electron-density maps: a simple optical device. , 1968, Journal of molecular biology.

[32]  J. Kraut,et al.  Crystallographic structure refinement of Chromatium high potential iron protein at two Angstroms resolution. , 1975, The Journal of biological chemistry.

[33]  G. Tollin,et al.  Kinetics and equilibria in partially reduced flavine solutions. , 1972, Biochemistry.

[34]  G. N. Ramachandran,et al.  Conformation of the LL and LD hairpin bends with internal hydrogen bonds in proteins and peptides. , 1973, Biochimica et biophysica acta.

[35]  R. Hardy,et al.  Isolation and characteristics of flavodoxin from nitrogen-fixing Clostridium pasteurianum. , 1966, The Journal of biological chemistry.

[36]  D. Cruickshank,et al.  The accuracy of electron-density maps in X-ray structure analysis , 1948 .

[37]  T. Steitz,et al.  The structure of carboxypeptidase AIII. Molecular structure at 6 resolution , 1966 .

[38]  C. J. Fritchie,et al.  Geometry of the unperturbed flavin nucleus. The crystal structure of 10‐methylisoalloxazine , 1973 .

[39]  P. Hemmerich,et al.  The chemical and electronic structure of the neutral flavin radical as revealed by electron spin resonance spectroscopy of chemically and isotopically substituted derivatives. , 1970, European journal of biochemistry.

[40]  Harold A. Scheraga,et al.  Calculations of Conformations of Polypeptides , 1968 .

[41]  E. Land,et al.  One-electron reactions in biochemical systems as studied by pulse radiolysis. 3. Ubiquinone. , 1970, The Journal of biological chemistry.

[42]  A. North,et al.  The combination of isomorphous replacement and anomalous scattering data in phase determination of non-centrosymmetric reflexions , 1965 .

[43]  R. M. Burnett,et al.  The structure of the oxidized form of clostridial flavodoxin at 1.9-A resolution. , 1974, The Journal of biological chemistry.

[44]  R. Kretsinger,et al.  Refinement of the structure of carp muscle calcium-binding parvalbumin by model building and difference Fourier analysis. , 1976, Journal of molecular biology.

[45]  R Diamond,et al.  Real-space refinement of the structure of hen egg-white lysozyme. , 1977, Journal of molecular biology.

[46]  C. Venkatachalam.  Stereochemical criteria for polypeptides and proteins. V. Conformation of a system of three linked peptide units , 1968, Biopolymers.

[47]  N. A. Sörensen,et al.  Studies on Flavin Derivatives. The Crystal Structure of 5-Acetyl-9-bromo-1,3,7,8,10-pentamethyl-1,5-dihydroisoalloxazine. , 1970 .

[48]  A. Rae.  The correlation of zones of X-ray intensity data from common reflexions , 1965 .

[49]  M. Yates.  Electron transport to nitrogenase in Azotobacter chroococcum: Azotobacter flavodoxin hydroquinone as an electron donor , 1972, FEBS letters.

[50]  M G Rossmann,et al.  A comparison of the structures of apo dogfish M4 lactate dehydrogenase and its ternary complexes. , 1977, Journal of molecular biology.

[51]  G. A. Jeffrey,et al.  The crystal structure of ribitol , 1969 .

[52]  J. E. McQueen,et al.  Computer manipulation of (macro)molecules with the method of local change , 1974 .

[53]  P. Song.  Electronic structure and photochemistry of flavins. IV. .sigma.-Electronic structure and the lowest triplet configuration of a flavin , 1968 .

[54]  L. Ingraham,et al.  A potentiometric study of the flavin semiquinone equilibrium. , 1968, Archives of biochemistry and biophysics.

[55]  G. G. Stokes.  "J." , 1890, The New Yale Book of Quotations.

[56]  S. Mayhew.  Studies on flavin binding in flavodoxins. , 1971, Biochimica et biophysica acta.

[57]  M. Ludwig,et al.  The structure of a clostridial flavodoxin. I. Crystallographic characterization of the oxidized and semiquinone forms. , 1969, The Journal of biological chemistry.

[58]  S. Ghisla,et al.  ROLE OF CHARGE‐TRANSFER INTERACTIONS IN FLAVOPROTEIN CATALYSIS , 1974 .

[59]  R. Smillie,et al.  Isolation of two proteins with chloroplast ferredoxin activity from a blue-green alga. , 1965, Biochemical and biophysical research communications.

[60]  C. J. Fritchie,et al.  The crystal structure of a riboflavin-metal complex. Ribofalavin silver perchlorate hemihydrate. , 1973, The Journal of biological chemistry.

[61]  L. Ingraham,et al.  The affinity of flavin semiquinones for certain aromatic compounds and disulfides. , 1970, Archives of biochemistry and biophysics.

[62]  L. Anderson.  Structures of deoxy and carbonmonoxy haemoglobin Kansas in the deoxy quaternary conformation. , 1975, Journal of molecular biology.

[63]  R. Dickerson,et al.  A least‐squares refinement method for isomorphous replacement , 1968 .

[64]  H. Scheraga,et al.  Energy parameters in polypeptides. VII. Geometric parameters, partial atomic charges, nonbonded interactions, hydrogen bond interactions, and intrinsic torsional potentials for the naturally occurring amino acids , 1975 .