Clostridium MP Research Articles

Tertiary structure of two‐electron reduced Megasphaera elsdenii flavodoxin and some implications, as determined by two‐dimensional 1H‐NMR and restrained molecular dynamics

The tertiary structure of the non-crystallizable two-electron-reduced Megasphaera elsdenii flavodoxin (15 kDa, 137 amino acid residues) has been determined using nuclear Overhauser enhancement restraints extracted from two-dimensional 1H-NMR spectra. A tertiary structure satisfying the experimental restraints very well (maximum NOE violation of 66 pm) was obtained with use of restrained molecular dynamics, using 509 distance restraints (including one non-NOE) on a starting structure modeled from the crystal structure of one-electron-reduced Clostridium MP flavodoxin. The protein consists of a central parallel beta-sheet surrounded on both sides by two alpha-helices. The flavin is positioned at the periphery of the molecule. The tertiary structure of the protein is highly defined with the exception of the flavin. The latter is expected to result from performing the restrained molecular dynamics simulation without water molecules and without proper charges on the flavin. The flavin, including the phosphate, the ribityl side chain and the isoalloxazine ring, is solvent accessible under the experimental conditions used and evidenced by a two-dimensional amide exchange experiment. This accessibility is expected to be important in the redox potential regulation of the semiquinone/hydroquinone couple of the protein. The amide exchange against deuterons and several typical line shapes in the two-dimensional NMR spectra are consistent with the structure generated. The structure is discussed in detail.

Secondary and tertiary structure characteristics of Megasphaera elsdenii flavodoxin in the reduced state as determined by two‐dimensional 1H NMR

The secondary structure of two-electron-reduced Megasphaera elsdenii flavodoxin has been determined by visual, qualitative inspection of the sequential connectivities involving C alpha H, C beta H and NH protons observed in NOESY (two-dimensional nuclear Overhauser enhancement spectroscopy) spectra. Results from an amide proton exchange experiment were used to confirm the secondary structure assignment and to demonstrate the compactness and stability of the protein. After the secondary structure elements were established, the global fold of the protein and the flavin binding site have been determined using nonsequential interresidual NOE connectivities as primary source of information. The secondary structure and the global fold of M. elsdenii and Clostridium MP flavodoxin appeared to be very similar, differences are observed however. M. elsdenii flavodoxin consists of a central parallel beta-sheet including five strands surrounded on both sides by a pair of alpha-helices.

A two‐dimensional 1H NMR study on Megasphaera elsdenii flavodoxin in the reduced state

Assignments for the 137 amino acid residues of Megasphaera elsdenii flavodoxin in the reduced state have been made using the sequential resonance assignment procedure. Several hydroxyl and sulfhydryl protons were observed at 41 degrees C at pH 8.3. Spin systems were sequentially assigned using phase-sensitive two-dimensional-correlated spectroscopy and phase-sensitive nuclear Overhauser enhancement spectroscopy. Spectra of the protein in H2O and of protein preparations either completely or partly exchanged against 2H2O were obtained. Use of the fast electron shuttle between the paramagnetic semiquinone and the diamagnetic hydroquinone state greatly simplified the NMR spectra, making it possible to assign easily the 1H resonances of amino acid residues located in the immediate neighbourhood of the isoalloxazine ring. The majority of the nuclear Overhauser effect contracts between the flavin and the apoprotein correspond to the crystal structure of the flavin domain of Clostridium MP flavodoxin, but differences are also observed. The assignments provide the basis for the structure determination of M. elsdenii flavodoxin in the reduced state as well as for assigning the resonances of the oxidized flavodoxin.

Chemical Synthesis and Expression of a Synthetic Gene for the Flavodoxin from Clostridium MP

A gene coding for the flavodoxin from Clostridium MP was designed, synthesized, and expressed in Escherichia coli. The sequence of the coding region was derived from the published amino acid sequence of the protein (Tanaka, M., Haniu, M., Yasunobu, K.T., and Mayhew, S. G. (1974) J. Biol. Chem. 249, 4393-4397) and was designed for optimal expression and for use of the cassette mutagenesis approach. The structural gene was subassembled in three sections, each of which was constructed by the enzymatic ligation of three complementary pairs of chemically synthesized oligodeoxyribonucleotides having short single-stranded ends complementary to that of the adjacent pair. Coligation of the three sections produced the final structural gene which consists of 420 nucleotides. The synthetic gene was cloned behind the hybrid tac promoter (Amman, E., Brosius, J., and Ptashne, M. (1983) Gene (Amst.) 25, 167-178) in the pKK223-3 vector or adjacent to the strong T7 RNA polymerase promoter in the pET-3a expression vector (Rosenberg, A.H., Lade, B. N., Chui, D-S., Lin, S-W., Dunn, J. J., and Studier, F. W. (1987) Gene (Amst.) 56, 125-135) for expression in E. coli. Upon induction with isopropyl-beta-D-thiogalactoside, the flavodoxin polypeptide was expressed from the artificial gene to levels approaching 20% of total extractable proteins using either expression system. The flavodoxin was purified from cellular extracts as the holoprotein containing bound flavin mononucleotide. The recombinant flavodoxin protein was found to have an ultraviolet/visible spectrum, amino-terminal sequence, and amino acid composition identical to the wild-type flavodoxin protein purified from Clostridium MP. This work represents the first chemical synthesis and expression in E. coli of an artificial gene coding for a bacterial flavodoxin.

Identification of Clostridium MP as Clostridium beijerinckii

Clostridium MP, an organism that has been widely used in st ructural and functional studies on the flavoprotein flavodoxin, has been identified as Clostridium beijerinckii.

The base sequence of the nifF gene of Klebsiella pneumoniae and homology of the predicted amino acid sequence of its protein product to other flavodoxins.

The nucleotide sequence of a 629 base-pair segment of DNA spanning the nifF gene of Klebsiella pneumoniae is presented. The structural gene comprises 531 base-pairs (175 codons, excluding the translational initiator and terminator) encoding an acidic polypeptide of 18950 Da. The nifF product thus belongs to the long-chain class of flavodoxins. It shows some sequence homology to the short-chain flavodoxins from Desulfovibrio vulgaris, Clostridium MP and Megasphaera elsdenii, and much stronger homology to long-chain flavodoxins from Azotobacter vinelandii and Anacystis nidulans. The long chain flavodoxins thus seem to constitute a well-conserved sub-group. The homology with the A. vinelandii flavodoxin is particularly strong, which may reflect their common function in nitrogen fixation.

The use of two-dimensional nuclear-magnetic-resonance spectroscopy and two-dimensional difference spectra in the elucidation of the active center of Megasphaera elsdenii flavodoxin.

1H-1H 'through bond' correlated (COSY) and 1H-1H 'through space' (NOESY) two-dimensional NMR techniques were applied to study the structure of Megasphaera elsdenii flavodoxin in the oxidized and reduced state. It is shown that two-dimensional NOESY difference spectra between spectra of flavodoxin in the reduced and semiquinone state are sensitive to the active center of the fully reduced state. The sphere of the active center observed in the difference spectra can be varied easily by changing the relative amount of flavodoxin semiquinone in the second sample. The difference NOESY spectra simplified the analysis of the complex spectra. Resonances could be assigned to Ala-56, Tyr-89 and Trp-91, which are located in the direct vicinity of the protein-bound flavin. The relative positions and side-chain dihedral angles of these residues are compared for the two redox states. Ala-56 and Tyr-89 show identical relative positions and dihedral angles in the two redox states, although the rotational motion of Tyr-89 is enhanced in the oxidized state. In both redox states Trp-91 is immobilized and extremely close to the prosthetic group. However, a small displacement of Trp-91 towards the (N(5) atom of the flavin occurs upon reduction. The results obtained for Trp-91 are in excellent agreement with crystallographic results of the related flavodoxin from Clostridium MP. However, the latter studies showed a somewhat different position of the tyrosine residue compared with our results.

On the mobility of riboflavin 5'-phosphate in Megasphaera elsdenii flavodoxin as studied by 13C-nuclear-magnetic-resonance relaxation.

The mobility of the isoalloxazine ring of the prosthetic group of Megasphaera elsdenii flavodoxin was investigated by a 13C relaxation study of the non-protonated ring atoms 2, 4, 4a and 10a. In this study a selectively enriched (greater than 90% 13C) prosthetic group was bound to the apoprotein. T1 and T2 values were determined at two magnetic field strengths, i.e. 8.46 T (90.5 MHz) and 5.88 T (62.8 MHz). Values of nuclear Overhauser effects (NOE) were determined at 5.88 T. It is shown that both the dipole-dipole interaction and the chemical shift anisotropy are important relaxation sources for all the carbon atoms investigated. The results are in agreement with a spectral density function of the isoalloxazine ring in which only the overall reorientational motion of the protein is accounted for. From this it is concluded that the isoalloxazine ring is tightly associated with the apoprotein. The protein-bound isoalloxazine ring does not exhibit large fluctuations on the nanosecond time scale, although small amplitude fluctuations cannot be excluded. This information was obtained by a combination of field-dependent T1 and NOE measurements. T2 values are in agreement with these results. On the basis of the dipolar part of the overall T1 values, the distance between the carbon investigated and the nearest proton was calculated and found to be in fair agreement with the crystallographic results of the related flavodoxin from Clostridium MP. In addition, it is shown that, based on the chemical shift anisotropy as a relaxation source, information on the internal mobility is difficult to obtain. The main reason for this is the low precision in the determination of the chemical shift anisotropy tensor.

Structure of oxidized flavodoxin from Anacystis nidulans

The structure of oxidized flavodoxin from the cyanobacterium Anacystis nidulans has been determined at 2.5 A resolution with phases calculated from ethylmercury phosphate and dimercuriacetate derivatives. The determination of partial sequences, including a total of 85 residues, has assisted in the interpretation of the electron density. Preliminary refinement of a partial model (1072 atoms) has reduced R to 0.349 for the 10.997 reflections between 2.0 and 5.0 A with 1 greater than 2 sigma. The polypeptide backbone, which comprises 167 residues in the current model, adopts the familiar beta-alpha-beta conformation found in other flavodoxins and in the nucleotide-binding domains of the pyridine-nucleotide dehydrogenases, with five parallel strands in the central sheet. Comparison with flavodoxin from Clostridium MP (138 residues) shows that extra residues of A. nidulans flavodoxin are accommodated in a major insertion about 20 residues in length, which forms a lobe adjacent to the fifth strand of parallel sheet, and in additions to several external segments. Residues added between the fourth sheet strand and the start of the third helix alter the environment of the pyrimidine end of the flavin mononucleotide ring. The flavin mononucleotide phosphate binds to the start of helix 1, interacting with hydroxyamino acids and with main-chain amide groups. Two hydrophobic residues, both tentatively identified as Trp, enclose the isoalloxazine ring; the solvent-exposed Trp is nearly parallel to the flavin ring. The hydrophobic environment provided by these residues must be partly responsible for the pronounced vibrational resolution of the flavin spectrum near 450 nm. The flavin ring is tilted relative to its orientation in Clostridium MP flavodoxin. In addition, atoms N-3 and O-2 alpha of the isoalloxazine appear to form hydrogen bonds to the backbone at CO97 and NH99 in a conformation entirely different from that found in Clostridium MP flavodoxin but structurally analogous to Desulfovibrio vulgaris flavodoxin.

A photo-CIDNP study of the active sites of Megasphaera elsdenii and Clostridium MP flavodoxins

Megasphaera elsdenii and Clostridium MP flavodoxins have been investigated by photo-CIDNP techniques. Using time-resolved spectroscopy and external dyes carrying different charges it was possible to assign unambiguously the resonance lines in the NMR-spectra to tyrosine, tryptophan and methionine residues in the two proteins. The results show that Trp-91 in M.elsdenii and Trp-90 in Cl.MP flavodoxin are strongly immobilized and placed directly above the benzene subnucleus of the prosthetic group. The data further indicate that the active sites of the two flavodoxins are extremely similar.

Structural and dynamic information on the complex of Megasphaera elsdenii apoflavodoxin and riboflavin 5'-phosphate. A phosphorus-31 nuclear magnetic resonance study.

It is shown that commercial FMN contains a considerable amount of the 4', 3', and 2' isomers and other phosphorus-containing compounds. These impurities can easily be analyzed and quantified by the 31P NMR technique. The phosphate group of FMN bound to Megasphaera elsdenii apoflavodoxin is probably in the dianionic form. Its chemical shift is almost independent of pH in the range 5.5-9.2 and of the redox state of the protein. The phosphate group of bound FMN is buried in the protein. Protons of the apoprotein and of bound water located in the vicinity of the phosphate group in native flavodoxin are not exchangeable with deuteron of the bulk solvent. These protons are, however, easily exchangeable in apoflavodoxin, as shown by reconstitution experiments. The distance between the phosphorus atom of bound FMN and the N(10) atom of the isoalloxazine moiety of FMN is calculated to be about 7.8 A. This result is in good agreement with X-ray data published for the related flavodoxin from Clostridium MP. The electron exchange between the oxidized and semiquinone state of M. elsdenii flavodoxin is rather slow (kexc' much less than 2 s-1) whereas that between the semiquinone and hydroquinone form is much more favored (kexc' much greater than 100 s-1). This indicates that the activation energy for the transition between the semiquinone and hydroquinone states must be smaller than that for the transition between the oxidized and semiquinone states. These results offer a reasonable explanation for the one-electron transfer reaction of flavodoxins in biological reactions.

MO Study of flavin–protein interactions in flavodoxin catalysis

AbstractMINDO/3 calculations have been performed on the Clostridium MP flavodoxin active site (a complex of the redox active coenzyme flavin mononucleotide sandwiched between the side chains of methionine and tryptophan) at various redox levels using coordinates derived from x‐ray diffraction studies of the holoenzyme. Frontier orbital indices were calculated and indicate that reduction of the flavin is accompanied by induced polar states in the amino acid side chains. This stabilization of charge by the amino acid side chains could account for the reaction rate enhancement of flavin reduction catalyzed by flavodoxin. Frontier orbitals for free flavin, for the flavodoxin bound flavin without the amino acid side chains, and for the oxidized Desulfovibrio vulgaris flavodoxin active site were computed for comparison.

Resonance coherent anti-Stokes Raman scattering spectra of oxidized and semiquinone forms of Clostridium MP flavodoxin.

Resonance coherent anti-Stokes Raman scattering spectra of oxidized and semiquinone forms of Clostridium MP flavodoxin.

One-electron reduction of flavodoxin. A fast kinetic study.

The reaction kinetics of fully oxidized flavodoxin from Clostridium MP with the hydrated electron have been investigated by the pulse radiolysis method. Four spectrally distinct processes have been observed with the ultimate formation of the singly reduced flavin form of the protein. The last two species obtained in the reaction sequence are spectrally similar, and are connected through a reaction which is first order. It is proposed that this reaction involves a protein conformational alteration.

Hydrogen bonding of flavoprotein. I. Effect of hydrogen bonding on electronic spectra of flavoprotein

The effect of hydrogen bonding on the transition energy and the oscillator strength of the isoalloxazine nucleus of flavins was studied by the molecular orbital method. Among the possible hydrogen bondings examined, characteristic spectral shifts were found for the hydrogen bondings at N(1) and N(5) of the nucleus. The hydrogen bonding at N(1) resulted in the shift of the first absorption band towards blue and that of the second one towards red. On the other hand, the hydrogen bonding at N(5) resulted in the shifts of both the first and the second band towards red. The spectral characteristics reported on Clostridium MP and Desulfovibrio vulgaris flavodoxin conicided with the calculated results. The application of the calculated results to d-amino acid oxidase ( d-amino acid: oxygen oxidoreductase (deaminating), EC 1.4.3.3) led to the conclusion that hydrogen bonding occurs at O(12), N(3)H, O(14) and N(5) of the isoalloxazine nucleus. The occurrence of hydrogen bondings at O(12), N(3)H, and O(14) is favorable for N(5) of the isoalloxazine nucleus to accept electron from an electron donor.

Structure of the semiquinone form of flavodoxin from Clostridium MP: Extension of 1.8 Å 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 semiquinono form were determined to 2.0 Å resolution by isomorphous replacement (〈 m 〉 = 0.725). Subsequently, the structure was refined at 1.8 Å 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 versus maps with Fourier coefficients of the form ( 2¦F o ¦− ¦F c ¦) exp (iα c ). To minimize bias in the fitting, groups of atoms were systematically omitted from the structure factors used in computation of the ( 2¦F o − ¦F c ¦ ) 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 Å 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 the oxidized agsemiquinone conversion ( R I = 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 a rigid body motion. The required transformation, determined for the isomorphous replacement maps by the method of Cox (1967), is equivalent to a screw motion with a rotation of 1.18 ° and a translation of −0.34 Å. The molecular structures of oxidized and semiquinone flavodoxins have been compared after superposition of models with idealized co-ordinates and discrepancy indices Rox = 0.213 and Rsq = 0.200. The root-mean-square distance between 523 backbone atoms (excluding sequences 56 to 59 and 89 to 91) is 0.308 Å.

Optical spectra and electronic structure of flavine mononucleotide in flavodoxin crystals.

The polarized single-crystal absorption spectra of the oxidized and semiquinone forms of flavodoxin from Clostridium MP have been measured with a double beam recording microspectrophotometer. The spectra establish that the radical species in the crystal is the neutral (blue) falvine semiquinone. Combination of the spectra reported here with polarization data from previous fluorescence and stretched-film studies provides transition moment directions for the first two phi-phi transitions of the oxidized form. Predictions of molecular orbital theory are in good agreement with these experimental directions. The crystal spectra of the semiquinone indicate that the two lowest frequency transitions have the same detailed orbital origin as the corresponding transitions of the oxidized form; in the semiquinone these transitions appear at lower frequency, are closer together, and, as predicted from detailed considerations of transition probabilities, exhibit approximately half the absorption intensity. Our hypothesis of a common orbital origin suggests that semiquinone formation takes place by the addition of an electron to the lowest empty phi orbital of the oxidized form without any gross electronic rearrangement.

The Structure of the Oxidized Form of Clostridial Flavodoxin at 1.9-A Resolution: DESCRIPTION OF THE FLAVIN MONONUCLEOTIDE BINDING SITE

Abstract An electron density map of the oxidized form of flavodoxin from Clostridium strain MP has been calculated to a resolution of 1.9 A employing isomorphous and anomalous scattering data from two heavy atom derivatives, Smiii and Aui. In the region from 3.0 to 1.9 A, the largest structure amplitudes (85% of the total | F | s) have been included in the computation. The over-all figure of merit is 0.808. The molecular model resulting from interpretation of this map has a chain length of 138 residues, in accord with the sequence (Tanaka, M., Haniu, M., Yasunobu, K. T., and Mayhew, S. G. (1974) J. Biol. Chem. 249, 4393–4396). As reported earlier, the molecule possesses a high degree of secondary structure, including a central parallel sheet and four helices. More than 70% of the residues appear to participate in backbone hydrogen bonding. Atomic coordinates, incorporating the sequence data, have been optimized by use of Diamond's model-building program. The flavin mononucleotide prosthetic group adopts a somewhat extended conformation. As expected for the oxidized state, the isoalloxazine ring is planar. It is partly shielded from solvent by a tryptophan residue and interacts on its inner surface with methionine. Protein atoms are also in appropriate positions for interaction with atoms N-1, O-2, and N-3 of the flavin ring, but N-5 appears too far removed to form a hydrogen bond with the apoprotein. The ribityl phosphate moiety also makes multiple contacts with protein groups. Two of the three O' oxygens are in positions where they may participate in hydrogen bonding; the phosphate is closely surrounded by four hydroxyamino acids and four backbone NH groups. However, the prosthetic group is accessible on one side to solvent. Peaks likely to represent solvent can be observed near the dimethylbenzene end of the isoalloxazine ring and adjacent to the 3'-ribityl oxygen. While the mutual arrangement of protein and ribityl phosphate is nearly identical with that found by Watenpaugh et al. (Watenpaugh, K. D., Sieker, L. C., Jensen, L. H., LeGall, J., and Dubourdieu, M. (1972) Proc. Nat. Acad. Sci. U. S. A. 69, 3185–3188) in Desulfovibrio vulgaris flavodoxin, the orientation and interactions of the flavin ring differ strikingly in the two flavodoxins. The structure of the oxidized form of Clostridium MP flavodoxin is very similar to that deduced earlier for the semiquinone form at 3.25 A, despite large differences in the x-ray intensities (Ri = 0.66).

The Amino Acid Sequence of the Clostridium MP Flavodoxin

Abstract The amino acid sequence of Clostridium strain MP flavodoxin was determined by partial use of manual and automated Edman degradation of S-β-carboxymethylcysteinylflavodoxin, of the cyanogen bromide fragments of the protein, of the tryptic peptides, and of the thermolysin-derived peptides of these starting materials. The sequence was determined to be: Met-Lys-Ile-Val-Tyr-Trp-Ser-Gly-Thr-Gly-Asn-Thr-Glu-Lys-Met-Ala-Glu-Leu-Ile-Ala-Lys-Gly-Ile-Ile-Glu-Ser-Gly-Lys-Asp-Val-Asn-Thr-Ile-Asn-Val-Ser-Asp-Val-Asn-Ile-Asp-Glu-Leu-Leu-Asn-Glu-Asp-Ile-Leu-Ile-Leu-Gly-Cys-Ser-Ala-Met-Gly-Asp-Glu-Val-Leu-Glu-Glu-Ser-Glu-Phe-Glu-Pro-Phe-Ile-Glu-Glu-Ile-Ser-Thr-Lys-Ile-Ser-Gly-Lys-Lys-Val-Ala-Leu-Phe-Gly-Ser-Tyr-Gly-Trp-Gly-Asp-Gly-Lys-Trp-Met-Arg-Asp-Phe-Glu-Glu-Arg-Met-Asn-Gly-Tyr-Gly-Cys-Val-Val-Val-Glu-Thr-Pro-Leu-Ile-Val-Gln-Asn-Glu-Pro-Asp-Glu-Ala-Glu-Gln-Asp-Cys-Ile-Glu-Phe-Gly-Lys-Lys-Ile-Ala-Asn-Ile-COOH. The sequence of this flavodoxin is of vital interest since it is required for the interpretation of crystal x-ray diffraction data at the 1.9 A level (Burnett, R. M., Darling, G. D., Kendall, D. S., LeQuesne, M. E., Mayhew, S. G., Smith, W., and Ludwig, M. L. (1974) J. Biol. Chem. 249, 4383–4392).

Structure of the radical form of clostridial flavodoxin: a new molecular model.

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-N10.