Studies on the incorporation of a covalently bound disubstituted phosphate residue into <i>Azotobacter vinelandii</i> flavodoxin <i>in vivo</i>

Boylan, Maire; Edmondson, Dale E.

doi:10.1042/bj2680745

Cited by 5 publications

(4 citation statements)

References 19 publications

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“…In addition, a 1.17 Å resolution structure of the flavodoxin purified from A. vinelandii established the underlying structural similarity to the recombinant protein reported previously . Although reported previously, no phosphodiester linkage between amino acid side chains was observed in this flavodoxin II isolated from A. vinelandii . This was the last unsolved structure of wildtype nitrogenase electron transfer proteins isolated from A. vinelandii , and allowed for the modeling of the interactions between the Fe‐protein and these proteins.…”

Section: Introductionsupporting

confidence: 78%

“…(A)] reflects the prototypical α/β‐architecture of long chain flavodoxins, including the insertion of 22‐amino acids in the fifth β‐strand, which is characteristic of long chain flavodoxins . No phosphodiester modification was observed in the structure, as has been reported previously to be present in A. vinelandii flavodoxin II; perhaps reflecting strain variations or differences in growth conditions. The Azotobacter flavodoxin II also has an insertion of eight amino acid residues (amino acids 64–71) near the FMN cofactor that is generally found in nitrogenase‐associated flavodoxins; these residues have been proposed to be involved in complex formation with the Fe‐protein of nitrogenase .…”

Section: Resultsmentioning

confidence: 68%

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Electrochemical and structural characterization of Azotobacter vinelandii flavodoxin II

et al. 2017

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Azotobacter vinelandii flavodoxin II serves as a physiological reductant of nitrogenase, the enzyme system mediating biological nitrogen fixation. Wildtype A. vinelandii flavodoxin II was electrochemically and crystallographically characterized to better understand the molecular basis for this functional role. The redox properties were monitored on surfactant‐modified basal plane graphite electrodes, with two distinct redox couples measured by cyclic voltammetry corresponding to reduction potentials of −483 ± 1 mV and −187 ± 9 mV (vs. NHE) in 50 mM potassium phosphate, 150 mM NaCl, pH 7.5. These redox potentials were assigned as the semiquinone/hydroquinone couple and the quinone/semiquinone couple, respectively. This study constitutes one of the first applications of surfactant‐modified basal plane graphite electrodes to characterize the redox properties of a flavodoxin, thus providing a novel electrochemical method to study this class of protein. The X‐ray crystal structure of the flavodoxin purified from A. vinelandii was solved at 1.17 Å resolution. With this structure, the native nitrogenase electron transfer proteins have all been structurally characterized. Docking studies indicate that a common binding site surrounding the Fe‐protein [4Fe:4S] cluster mediates complex formation with the redox partners Mo‐Fe protein, ferredoxin I, and flavodoxin II. This model supports a mechanistic hypothesis that electron transfer reactions between the Fe‐protein and its redox partners are mutually exclusive.

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Section: Introductionsupporting

confidence: 78%

Section: Resultsmentioning

confidence: 68%

Electrochemical and structural characterization of Azotobacter vinelandii flavodoxin II

et al. 2017

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“…k" is 2.1 20.7 10 ' cinis for wild-type, C69A, and C69S flavo- The solid line is a least-squares fit to the data assuming a model with three redox-linked pK valucs: pK,,,,,, = 5.39 iO.08, pK,,, = 7.29 20.14, pK,,,,,, = 7.84?0.14. Data points with error bars were not included in thc fit. The estimated standard deviations for other data points are in the range [3][4][5][6][7][8][9] mV. Experimental conditions were as described in the Experimental Procedures section.…”

Section: Resultsmentioning

confidence: 99%

Redox Properties of Wild‐Type, Cys69Ala, and Cys69Ser Azotobacter Vinelandii Flavodoxin II as Measured by Cyclic Voltammetry and EPR Spectroscopy

Steensma

Heering

Hagen

et al. 1996

European Journal of Biochemistry

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This study deals with the detailed electrochemistry and complete EPR-monitored titrations of flavodoxin I1 of Azotobacter vinelandii (ATCC 478). Since wild-type flavodoxin dimerises via intermolecular disulphide bond formation between Cys69 residues, Cys69 has been replaced by both an alanine and a serine residue. Redox properties of the C69A and C69S flavodoxin mutants were compared to those of wild-type flavodoxin. In the presence of the promotor neomycin, C69A and C69S flavodoxin showed a reversible response of the semiquinone/hydroquinone couple at the glassy carbon electrode. However, the addition of dithiothreitol proved to be necessary for the stabilisation of the wild-type flavodoxin response. EPR-monitored redox titrations of wild-type and C69A flavodoxin at high and low pH confirmed the redox potentials measured using cyclic voltammetry. The pH dependence of the semiquinonelhydroquinone redox potentials cannot be described using a model assuming one redox-linked pK. Instead, the presence of at least two redox-linked protonation sites is suggested: pKrcd,l = 5.3920.08, pK,,, = 7.29t0.14, and pK,,,,, = 7.8450.14 with E,,,,7 = -4 5 9 t 4 mV, and a constant redox potential at high pH of -485 2 4 rnV. The dependence of the semiquinonelhydsoquinone redox potential on temperature is -0.5 20.1 mV . K-', yielding AH" = 28.6i.1.5 kJ . mol-' and AS" = -50.0t6.2 J . mol-I . K-'. No significant differences in redox properties of wild-type, C69A, and C69S flavodoxin were observed. The electrochemical data suggest that replacement of Cys69 in the vicinity of the FMN by either an alanine or a serine residue does not alter the dielectric properties and structure of A. vinelnndii flavodoxin 11.Keywords : flavodoxins ; redox potential ; cysteine mutants ; bioelectrochemistry ; EPR.Flavodoxins are able to function as electron carriers between redox proteins because they contain a non-covalently bound FMN molecule [I]. The FMN molecule can exist in three redox states : oxidised flavoquinone, one-electron reduced flavosemiquinone and two-electron reduced flavohydroquinone. Reported midpoint potentials for the quinonekemiquinone couple (EJ and the semiquinone/hydroquinone couple ( E l ) of free FMN at pH 7 are E, = -238 mV and El = -172 mV 121, and E, = -314 mV and E l = -124 mV 131. Due to the rapid disproportionation of the semiquinone state, free FMN in solution cycles between the completely oxidised and the two-electron reduced state 141. The binding of FMN to flavodoxins considerably increases the stability of the semiquinone state and alters the redox potentials E, and E l : the midpoint potential for the quinonel semiquinone couple (E,) is increased, whereas the midpoint potential for the semiquinone/hydroquinone couple (El) is decreased. In most low-potential oxidation-reduction reactions, FMN in flavodoxins only cycles between the semiquinone and the hydroquinone state [I].Correspondence to C. P. M. van Mierlo, Department of Biochemistry, Wagcningen Agricultural University, Dreijenlaan 3, NL-6703 HA Wageningen, The ...

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“…to be a bisubstituted phosphate residue involving serine and threonine [15]. Phosphate was incorporated into newly synthesized flavodoxin in i o under both N # -fixing and NH % + -sufficient conditions and this modification has been suggested to have a structural role [16]. The other phosphate residue (δ l k140.8 p.p.m.)…”

Section: P-nmr Measurements Of Purified Avfld 1 and Avfldmentioning

confidence: 99%

Flavodoxin 1 of Azotobacter vinelandii: characterization and role in electron donation to purified assimilatory nitrate reductase

Gangeswaran

Eady

1996

Biochemical Journal

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Flavodoxins synthesized by Azotobacter vinelandii strain UW 36 during growth on nitrate as nitrogen source were separated by FPLC on a Mono Q column into two species, flavodoxin 1 (AvFld 1) and flavodoxin 2 (AvFld 2). Both proteins migrated as single bands on SDS/PAGE. AvFld 1 was approx. 5-fold more abundant than AvFld 2 in the unresolved flavodoxin mixture. N-terminal amino acid analysis showed the sequence of AvFld 2 to correspond to the nif F gene product, an electron donor to nitrogenase. The sequences also show that these species corresponded to the flavodoxins Fld A and Fld B isolated from N2-grown cultures of the closely related organism Azotobacter throococcum [Bagby, Barker, Hill, Eady and Thorneley (1991) Biochem.J.277, 313-319]. Electrospray mass spectrometry gave M, values for the polypeptides of 19430 +/- 3 and 19533 +/- 5 respectively. 31P-NMR measurements showed that in addition to the phosphate associated with the FMN (delta = -136.3 p.p.m. and -135.48 p.p.m.), AvFld 1 had a signal at delta = -142.1 p.p.m. and AvFld 2 at delta = -138.59 p.p.m. present in substoichiometric amounts with FMN. These appeared to arise from unstable species since they were readily lost on further manipulation of the proteins. The mid-point potentials of the semiquinone hydroquinone redox couples were -330 mV and -493 mV for AvFld 1 and AvFld 2 respectively, but only AvFld 1 was competent in donating electrons to the purified assimilatory nitrate reductase of A. vinelandii to catalyse the reduction of nitrate to nitrite. Flavodoxin isolated from NH4(+)-grown cells (Fld 3) also functioned as electron donor at half the rate of AvFld 1, but ferredoxin 1 from A. chroococcum did not.

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Studies on the incorporation of a covalently bound disubstituted phosphate residue into Azotobacter vinelandii flavodoxin in vivo

Cited by 5 publications

References 19 publications

Electrochemical and structural characterization of Azotobacter vinelandii flavodoxin II

Electrochemical and structural characterization of Azotobacter vinelandii flavodoxin II

Redox Properties of Wild‐Type, Cys69Ala, and Cys69Ser Azotobacter Vinelandii Flavodoxin II as Measured by Cyclic Voltammetry and EPR Spectroscopy

Flavodoxin 1 of Azotobacter vinelandii: characterization and role in electron donation to purified assimilatory nitrate reductase

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