The Structure of a Clostridial Flavodoxin

Ludwig, Martha; Andersen, Robert D.; Mayhew, Stephen G.; Massey, Vincent

doi:10.1016/s0021-9258(18)63579-3

Cited by 38 publications

(16 citation statements)

References 8 publications

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“…Apoprotein was added to excess 1-deaza-FMN. After separation of free 1-deaza-FMN and concentration of the protein by centrifugation through a Centricon 10 microconcentrator (Amicon), samples were prepared for crystallization by mixing protein (final concentration 5 mg/mL) with varying amounts of 2.6 M ammonium sulfate and 0.2 M phosphate at pH 6.8 (Ludwig et al, 1969). Crystallization droplets were equilibrated with ammonium sulfate at concentrations of 2.0-2.4 M. The resulting crystals displayed a space group which was identical with that of native flavodoxin, P3]21, with slightly different cell dimensions: a Diffraction data were collected from one crystal at 4 °C by using a Xuong-Hamlin dual area detector system, with crystal-detector distances of 607 and 551 mm.…”

Section: Methodsmentioning

confidence: 99%

Structure and oxidation-reduction behavior of 1-deaza-FMN flavodoxins: modulation of redox potentials in flavodoxins

Ludwig¹,

Schopfer²,

Metzger³

et al. 1990

Biochemistry

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Flavodoxins from Clostridium beijerinckii and from Megasphaera elsdenii with 1-carba-1-deaza-FMN substituted for FMN have been used to study flavin-protein interactions in flavodoxins. The oxidized 1-deaza analogue of FMN binds to apoflavodoxins from M. elsdenii and C. beijerinckii (a.k.a. Clostridium MP) with association constants (Ka) of 1.0 x 10(7) M-1 and 3.1 x 10(6) M-1, values about 10(2) less than the corresponding Ka values for FMN. X-ray structure analysis of oxidized 1-deaza-FMN flavodoxin from C. beijerinckii at 2.5-A resolution shows that the analogue binds with the flavin atoms in the same locations as their equivalents in FMN but that the protein moves in the vicinity of Gly 89 to accommodate the 1-CH group, undergoing displacements which increase the distance between position 1 of the flavin ring and the main-chain atoms of Gly 89 and move the peptide hydrogen of Gly 89 by about 0.6 A. The X-ray analysis implies that protonation of normal flavin at N(1), as would occur in formation of the neutral fully reduced species, would result in a similar structural perturbation. The oxidation-reduction potentials of 1-deaza-FMN flavodoxin from M. elsdenii have been determined in the pH range 4.5-9.2. The oxidized/semiquinone equilibrium (E'0 = -160 mV at pH 7.0) displays a pH dependence of -60 mV per pH unit; the semiquinone/reduced equilibrium (E'0 = -400 mV at pH 7.0) displays a pH dependence of -60 mV per pH unit at low pH and is pH independent at high pH, with a redox-linked pK of 7.4. Spectral changes of fully reduced 1-deaza-FMN flavodoxin with pH suggest that this latter pK corresponds to protonation of the flavin ring system (the pK of free reduced 1-deaza-FMN is 5.6 [Spencer, R., Fisher, J., & Walsh, C. (1977) Biochemistry 16, 3586-3593]. The pK of reduced 1-deaza-FMN flavodoxin provides an estimate of the electrostatic interaction between the protein and the bound prosthetic group; the free energy of binding neutral reduced 1-deaza-FMN is more negative than that for binding the anionic reduced 1-deaza-FMN by 2.4 kcal.(ABSTRACT TRUNCATED AT 250 WORDS)

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Section: Methodsmentioning

confidence: 99%

Structure and oxidation-reduction behavior of 1-deaza-FMN flavodoxins: modulation of redox potentials in flavodoxins

Ludwig¹,

Schopfer²,

Metzger³

et al. 1990

Biochemistry

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“…Enzyme-cofactor hydrogen bond interactions involving carbonyl oxygens O(2) and O(4), and imide proton N(3)H of flavin are a prevalent motif in flavoproteins, 25,26 and have been postulated to play an important role in flavin redox modulation. 27 Since this hydrogen bonding predominantly involves protein backbone functionality (Figure 2), the effectiveness of mutation studies in probing the role of these interactions on the redox behavior of the cofactor is limited.…”

Section: Redox Modulation Through Hydrogen Bondingmentioning

confidence: 99%

“…Aromatic stacking interactions are important features in a number of flavoproteins such as the flavodoxins, 26 in which the two faces of the FMN cofactor are flanked by either a tryptophane/tyrosine or a tryptophane/methionine dyad. These electron-transfer proteins utilize exclusively the Fl rad H/Fl red Hredox cycle, facilitated by selective stabilization of the neutral radical.…”

Section: Redox Modulation Through π-Stackingmentioning

confidence: 99%

“…Another frequently observed molecular recognition motif in flavoproteins is the proximity of electron-rich donor atoms such as oxygen (tyrosyl hydroxyl group, 40 backbone carbonyl 45 ) or sulfur (methionine thioether, 26 cysteine thiol, or cystine disulfide 25 ) to the face of the flavin cofactor. 46 To probe these interactions, we again exploited the preorganized binding geometry provided by the modular xanthene receptors.…”

Section: Redox Modulation Through Donor Atom-π Interactionsmentioning

confidence: 99%

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From Enzyme to Molecular Device. Exploring the Interdependence of Redox and Molecular Recognition

1998

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Angelika Niemz was born in Rheinfelden, Germany, in 1970. After receiving her B.Sc. in chemistry at the University of Konstanz in 1993, she came to the United States through the Baden Wu ¨rttemberg-Massachusetts exchange program. Currently a Ph.D. student in the Rotello group at the University of Massachusetts, her interests span the areas of biological, organic, and physical chemistry. Her current research focuses on the characterization of host-guest systems using an array of magnetic resonance, electrochemical, spectroelectrochemical, and surface characterization methods. Vincent M. Rotello received his B.S. from the Illinois Institute of Technology in 1985. He obtained a Ph.D. with Harry Wasserman from Yale University in 1990, and then did an NSF Postdoctoral Fellowship with Julius Rebek at MIT. He is currently an Associate Professor in the Department of Chemistry and the Program in Molecular and Cellular Biology at the University of Massachusetts. His principle motivation in research is the fundamental understanding of noncovalent interactions, and the application of this knowledge to biological and materials issues. Scheme 1. Common Redox and Protonation States of the Flavin Cofactor (lumiflavin, R ) CH 3 ; FMN, R ) CH 2 ((CHOH) 4 -phosphate; FAD, R ) CH 2 ((CHOH) 4 -pyrophosphate-adenosine; flavin 1, R ) CH 2 CH(CH 3 ) 2 )

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“…On binding to the apoflavodoxin, the midpoint redox potentials of the FMN are drastically altered, and the FMN semiquinone becomes much more stable. This allows flavodoxin to behave as a one-electron transfer center, that, in vivo, cycles between the semiquinone and the fully reduced forms (Ludwig and Luschinsky, 1992;Mayhew and Tollin, 1992). The three-dimensional structures of several flavodoxins are known (Watenpaugh et al, 1973;Burnett et al, 1974;Smith et al, 1983;Fukuyama et al, 1990;van Mierlo et al, 1990;Rao et al, 1992;Genzor et al, 1996b), and the x-ray crystal structure of Desulfovibrio vulgaris flavodoxin substituted with riboflavin has also been determined (Walsh et al, 1998).…”

Section: Introductionmentioning

confidence: 99%

Electron-Nuclear Double Resonance and Hyperfine Sublevel Correlation Spectroscopic Studies of Flavodoxin Mutants from Anabaena sp. PCC 7119

Medina

Lostao

Sancho

et al. 1999

Biophysical Journal

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The influence of the amino acid residues surrounding the flavin ring in the flavodoxin of the cyanobacterium Anabaena PCC 7119 on the electron spin density distribution of the flavin semiquinone was examined in mutants of the key residues Trp(57) and Tyr(94) at the FMN binding site. Neutral semiquinone radicals of the proteins were obtained by photoreduction and examined by electron-nuclear double resonance (ENDOR) and hyperfine sublevel correlation (HYSCORE) spectroscopies. Significant differences in electron density distribution were observed in the flavodoxin mutants Trp(57) --> Ala and Tyr(94) --> Ala. The results indicate that the presence of a bulky residue (either aromatic or aliphatic) at position 57, as compared with an alanine, decreases the electron spin density in the nuclei of the benzene flavin ring, whereas an aromatic residue at position 94 increases the electron spin density at positions N(5) and C(6) of the flavin ring. The influence of the FMN ribityl and phosphate on the flavin semiquinone was determined by reconstituting apoflavodoxin samples with riboflavin and with lumiflavin. The coupling parameters of the different nuclei of the isoalloxazine group, as detected by ENDOR and HYSCORE, were very similar to those of the native flavodoxin. This indicates that the protein conformation around the flavin ring and the electron density distribution in the semiquinone form are not influenced by the phosphate and the ribityl of FMN.

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The Structure of a Clostridial Flavodoxin

Cited by 38 publications

References 8 publications

Structure and oxidation-reduction behavior of 1-deaza-FMN flavodoxins: modulation of redox potentials in flavodoxins

Structure and oxidation-reduction behavior of 1-deaza-FMN flavodoxins: modulation of redox potentials in flavodoxins

From Enzyme to Molecular Device. Exploring the Interdependence of Redox and Molecular Recognition

Electron-Nuclear Double Resonance and Hyperfine Sublevel Correlation Spectroscopic Studies of Flavodoxin Mutants from Anabaena sp. PCC 7119

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