The Structure of a Clostridial Flavodoxin, an Electron-transferring Flavoprotein. III. An Interpretation of an Electron-Density Map at a Nominal Resolution of 3.25 A

116

Flavodoxin from Desulfovibrio vulgaris crystallizes in the oxidized form as well-formed, tetragonal bipyramids, space group P43212, unit-cell parameters, a = b = 51.6 A, c = 139.6 A, 8 molecules per unit cell.The structure has been determined at 2.5-A resolution with phases based on a single isomorphous derivative. The phase ambiguity of a single derivative was resolved by use of anomalous scattering from the single-site Sm+3.The molecule has a five-strand pleated sheet core with two long helices on either side of the sheet. The flavin mononucleotide lies mostly buried on one side of the molecule, but the methyl groups, one edge of the flavin, and part of the ribityl are exposed at the surface.Originally, the name flavodoxin was given by Knight et al.(1) to a small flavoprotein found in extracts of Clostridium pasteurianum grown in iron-deficient medium. The protein was able to replace ferredoxin as an electron carrier (2, 3).After this discovery, other organisms have been shown to contain a similar flavoprotein: Desulfovibrio gigas (4), Peptostreptococcus elsdenii (5), Desulfovibrio vulgaris (6), Clostridium .vIP (7), Rhodospirillum rubrum (8), Chlorella fusca (9), and Escherichia coli (10).Flavodoxins have been defined as low molecular weight proteins with one flavin mononucleotide prosthetic group per molecule and having the ability to function interchangeably with ferredoxin in photosynthetic NADDP+ reduction (11).In Desulfovibrio, flavodoxin can replace ferredoxin in the reduction of sulfite and other sulfur compounds (4, 12, 13), and in the formation of hydrogen from pyruvate via the phosphoroclastic reaction (14).When thiosulfate is reduced by molecular hydrogen in the presence of Desulfovibrio extracts that have been depleted of flavodoxin, ferredoxin, and etochrome cc3, any one of these three electron carriers can couple the reaction (13,15 Sm+3 than for Yb+3, the Sm3+ derivative was used in this work, and to this point only about 50 mg of the protein has been required. Data collectionData were collected on a four-circle, computer-controlled diffractometer with a modified version of the control program of Lenhert and Henry (17). Ni-filtered CuKa radiation was used with a focal spot 0.4 X 10 mm and a take-off angle set at 6°. For both the native crystal and the Sm3+-derivative crystal, data were collected in the same way by a five-step w/20 scan across the top of each reflection. Each step was 0.080 in 20.Backgrounds were not measured for the individual reflections. Instead, the background was determined as a function of 20 by collecting counts at many points between reflections and assuming it to be independent of direction in space. The step scan used to collect data and the method of approximating the background was made necessary by the long c axis.Friedel-related reflections were collected by alternately measuring 32 reflections at +20, followed by 32 reflections at -20. The groups were staggered, however, so that the reflections of each Friedel pair were collected 16 reflections apart.

Section: Location Of Heavy Atoms and Phasing Of The Datamentioning

confidence: 99%

Structure of the Oxidized Form of a Flavodoxin at 2.5-Å Resolution: Resolution of the Phase Ambiguity by Anomalous Scattering

Watenpaugh

Sieker

Jensen

et al. 1972

116

“…The prosthetic group is similarly situated in both proteins, but the flavin mononucleotide-protein interactions appear to differ in certain respects. Some differences are not unexpected in view of the known dissimilarities in amino-acid composition (3,6), in optical and circular dichroic spectra (7), and in affin'ty for modified flavins (7,8 Two loops of polypeptide chain, one near residue 60 and the other near residue 90, interact with the flavin ring. The loop near residue 60 contributes a large residue in contact with the inner face of the isoalloxazine moiety; the loop near residue 90 contains an aromatic residue, probably tyrosine, stacked approximately parallel to the flavin mononucleotide ring on the "solvent side" of the prosthetic group (Fig.…”

mentioning

confidence: 99%

“…Earlier we reported an interpretation of an electron-density map of Clostridium MP flavodoxin semiquinone at 3.25-A resolution. The model constructed on the basis of that map was considered tentative in several regions, and the orientation of flavin mononucleotide could not be assigned unequivocally (3).…”

mentioning

confidence: 99%

Structure of the Radical Form of Clostridial Flavodoxin: A New Molecular Model

Andersen

Apgar

Burnett

et al. 1972

Self Cite

Interpretation of a new electron-density map at 3.25-A resolution has led to a somewhat revised model for the free radical (semiquinone) structure of flavodoxin from Clostridium MP. Although the general conformation of the molecule is very similar to that of oxidized Desulfovibrio vulgaris flavodoxin, flavin mononucleotide-protein interactions are not identical in the two flavodoxins. In the Cl. MP semiquinone molecule, the isoalloxazine ring appears to retain the essentially planar conformation characteristic of oxidized flavins; within the limits imposed by the resolution of the data, the map shows no evidence for bending of the isoalloxazine ring about N5-N1O.Analysis of the structure of flavodoxin crystals is expected to provide a detailed picture of flavin mononucleotide-protein interactions, which account for the characteristic chemical properties of these model flavoproteins (1, 2). Earlier we reported an interpretation of an electron-density map of Clostridium MP flavodoxin semiquinone at 3.25-A resolution. The model constructed on the basis of that map was considered tentative in several regions, and the orientation of flavin mononucleotide could not be assigned unequivocally (3).Recently, we calculated another map of the semiquinone form of flavodoxin at 3.25-A resolution, using three heavyatom derivatives, Sm+3, Pt+2, and Au+', and giving more weight to anomalous scattering differences in the phasing. The resulting map had a much improved figure of merit, 0.80, and has proven easier to interpret. The new map clearly suggested a flavin mononucleotide position different from that proposed earlier and required some revisions in the chain tracing ( Fig. 1). At about the same time, Watenpaugh et al. (4) succeeded in determining the structure of the oxidized form of a very similar protein from Desulfovibrio vulgaris at 2.5-A resolution. Part of our current model of the semiquinone form was constructed after a preliminary sketch of the model of Watenpaugh et al. had been kindly sent to us by Dr. Jensen. We describe our new molecular model briefly here in order to facilitate comparison of the two structures (5).Despite the greater chain length of flavodoxin from D.vulgaris (about 10 additional residues), the three-dimensional structures of the two molecules are clearly homologous. The prosthetic group is similarly situated in both proteins, but the flavin mononucleotide-protein interactions appear to differ in certain respects. Some differences are not unexpected in view of the known dissimilarities in amino-acid composition (3, 6), in optical and circular dichroic spectra (7), and in affin'ty for modified flavins (7,8 Fig. 1. In the matching regions, the average displacement of a total of 113 alpha carbons in constructing the new model was 1.7 A; in the best-defined helix, HI, the change was only 0.5 A. However, altered chain connections in the vicinity of the Nterminus and the flavin mononucleotide have led to a quite different ordering of the amino-acid sequence along the model. In the interpretati...

“…Comparison of the diffraction patterns from crystals of Cl. MP flavodoxin in the semiquinone and fully reduced states leads to the con-Biochemistry: James et al clusion that the semiquinone and reduced structures must be nearly identical (24). Furthermore, electron density maps of the flavin region show the conformations of the oxidized and radical forms to be very similar (15).…”

Section: Resultsmentioning

confidence: 99%

Dependence of the Proton Magnetic Resonance Spectra on the Oxidation State of Flavodoxin from Clostridium MP and from Peptostreptococcus elsdenii

James

Ludwig

Cohn

1973

The broadening of protein nuclear magnetic resonances in the spectra of the semiquinone forms of flavodoxins derived from Clostridium MP and Peptostreptococcus elsdenii relative to the resonances in the oxidized and reduced forms is highly selective. Spectra from both species of flavodoxin indicate that conformational differences between the oxidized and fully reduced states are minor and, consequently, the broadening in the semiquinone form is ascribed to the paramagnetic effect of the flavin free radical. The chemical shifts of the paramagnetically broadened lines are used in conjunction with x-ray crystallographic models to assign peaks to aminoacid residues in the proximity of the flavin mononucleotide. Species-dependent differences in the spectra can generally be attributed to differences in amino-acid composition and sequence. The spectra from both species of flavodoxin indicate that there is slow exchange between oxidized and semiquinone forms or reduced and semiquinone forms of the flavodoxins with a limit of kex < 50 sec'I for the exchange rate.Flavodoxins are small flavoproteins (molecular weight 15,000-22,000) that replace ferredoxins as low-potential electron carriers. Several organisms, including anaerobes (1-5), photosynthetic bacteria (6), blue-green (7, 8) and eukaryotic algae (9), nitrogen-fixing aerobes (10, 11), and Escherichia coli (12), synthesize flavodoxins. The purified proteins all contain one flavin mononucleotide per molecule, with no other prosthetic groups or bound metal ions.Flavodoxins do not react directly with pyridine nucleotides or other oxidizable substrates but rather act as substrates for other proteins. The oxidation-reduction potential for the oneelectron reduction of the semiquinone radical form is about -0.4 V (13), in the range typical for ferredoxins. In flavodoxins derived from Peptostreptococcus elsdenii and Clostridium MP, the potentials of the two one-electron steps are separated by more than 0.2 V (5, 13) so that the oxidized, semiquinone, and fully reduced forms of the protein are each accessible for chemical and structural studies. Recent x-ray studies have shown that the three-dimensional structures of flavodoxins from Desulfovibrio vulgaris (14) and Cl. MP (15) are very similar in spite of the differences in composition and the greater chain length (about 10 residues) of the D. vulgaris protein. In both structures the isoalloxazine ring is bound near the surface, but the flavin-protein interactions differ somewhat in the two species.In this paper, the proton nuclear magnetic resonance (NMR) spectra of flavodoxins from P. elsdenii and Cl. MP in each of the three oxidation states were compared. The phenomena observable in the present investigation differ from previous NMR investigations of iron-containing electron carriers (16, 17) in two essential ways: (1) three oxidation states of flavodoxins are available for comparison, two diamagnetic species, the oxidized and fully reduced forms, and one paramagnetic species, the semiquinone form; (2) the dominant...