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

Abstract

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.

Primary sequence, oxidation‐reduction potentials and tertiary‐structure prediction of Desulfovibrio desulfuricans ATCC 27774 flavodoxin

“…After these two steps the constraints are removed both from the mainchain and the Fh4N-side-chain atoms. The atoms of the isoalloxazine ring were kept rigid and always coplanar, in agreement with X-ray and NMR observations on flavodoxins 7. Tridimensional similarity between Desulfovibrio species flavodoxins based on the D. vulgaris structure.…”

“…6). [2,[4][5][6][7][8][42][43][44]. The third and fourth steps were performed considering the same type of interactions used in the first and second steps, respectively.…”

Fluorescence and optical characteristics of reduced flavines and flavoproteins

“…It is therefore reasonable to assume that the occurrence of fluorescence emission in (oxidized as well as reduced) flavoproteins is conditioned by the presence of specific amino acids close to the flavine binding site and also to the presence of specific (quenching) hydrogen bridges between protein and coenzyme. In agreement with this assumption a tryptophan and a methionine group in flavodoxin from Clostridium MP (Anderson ef al., 1972 (Watenpaugh et al, 1972) have been shown to be located in Van der Waals contact with the coenzyme by X-ray crystallography. It should be pointed out, however, that, due to the "opposite" chemical and electronic properties of FI,, (electron deficient, acceptor) and Fl,,d (electron rich, donor), different interactions should be expected at the two oxidation levels.…”

8α‐Substituted flavins of biological importance

“…The spectrum of the peptic peptide is quite similar in shape to those of the flavodoxins [57,58]. Recent X-ray crystallographic studies of two flavodoxins [59,60] reveal the presence of a tyrosyl residue in a planar 'stacking' arrangement with the flavin. Thus, it may be concluded that in the case of the peptic peptide the N-terminal tyrosyl residue interacts with the flavin ring in a parallel 'stacking' manner.…”