A modified flavodoxin with altered redox potentials is less efficient in electron transfer to nitrogenase

The flavodoxins of the Azotobacter vinelandii wild-type and a mutant strain TZN 200 have been studied. Although the primary structure of the two proteins is the same, the ability of the mutant flavodoxin to donate electrons to nitrogenase is reduced by 75%. One reason may be the raised mid-point potential of -435 mV for the semiquinone/hydroquinone couple in the mutant flavodoxin. The respective redox potential for the wild-type flavodoxin was found to be -480 mV. As shown by paper chromatography and light absorption spectroscopy, the structure of FMN is modified in the TZN 200 flavodoxin.     

Oxidation-reduction potentials of ferredoxin-NADP+ reductase and flavodoxin from Anabaena PCC 7119 and their electrostatic and covalent complexes.

The oxidation-reduction potentials of ferredoxin-NADP+ reductase and flavodoxin from the cyanobacterium Anabaena PCC 7119 were determined by potentiometry. The potentials at pH 7 for the oxidized flavodoxin/flavodoxin semiquinone couple (E2) and the flavodoxin semiquinone/hydroquinone couple (E1) were -212 mV and -436 mV, respectively. E1 was independent of pH above about pH 7, but changed by approximately -60 mV/pH below about pH 6, suggesting that the fully reduced protein has a redox-linked pKa at about 6.1, similar to those of certain other flavodoxins. E2 varied by -50 mV/pH in the range pH 5-8. The redox potential for the two-electron reduction of ferredoxin-NADP+ reductase was -344 mV at pH 7 (delta Em = -30 mV/pH). In the 1:1 electrostatic complex of the two proteins titrated at pH 7, E2 was shifted by +8 mV and E1 was shifted by -25 mV; the shift in potential for the reductase was +4 mV. The potentials again shifted following treatment of the electrostatic complex with a carbodiimide, to covalently link the two proteins. By comparison with the separate proteins at pH 7, E2 for flavodoxin shifted by -21 mV and E1 shifted by +20 mV; the reductase potential shifted by +2 mV. The potentials of the proteins in the electrostatic and covalent complexes showed similar pH dependencies to those of the individual proteins. Qualitatively similar changes occurred when ferredoxin-NADP+ reductase from Anabaena variabilis was complexed with flavodoxin from Azotobacter vinelandii. The shifts in redox potential for the complexes were used with previously determined values for the dissociation constant (Kd) of the electrostatic complex of the two oxidised proteins, in order to estimate Kd values for the interaction of the different redox forms of the proteins. The calculations showed that the electrostatic complexes, formed when the proteins differ in their redox states, are stronger than those formed when both proteins are fully oxidized or fully reduced.     

Redox and flavin-binding properties of recombinant flavodoxin from Desulfovibrio vulgaris (Hildenborough).

Flavodoxin from Desulfovibrio vulgaris (Hildenborough) has been expressed at a high level (3-4% soluble protein) in Escherichia coli by subcloning a minimal insert carrying the gene behind the tac promoter of plasmid pDK6. The recombinant protein was readily isolated and its properties were shown to be identical to those of the wild-type protein obtained directly from D. vulgaris, with the exception that the recombinant protein lacks the N-terminal methionine residue. Detailed measurements of the redox potentials of this flavodoxin are reported for the first time. The redox potential, E2, for the couple oxidized flavodoxin/flavodoxin semiquinone at pH 7.0 is -143 mV (25 degrees C), while the value for the flavodoxin semiquinone/flavodoxin hydroquinone couple (E1) at the same pH is -440 mV. The effects of pH on the observed potentials were examined; E2 varies linearly with pH (slope = -59 mV), while E1 is independent of pH at high pH values, but below pH 7.5 the potential becomes less negative with decreasing pH, indicating a redox-linked protonation of the flavodoxin hydroquinone. D. vulgaris apoflavodoxin binds FMN very tightly, with a value of 0.24 nM for the dissociation constant (Kd) at pH 7.0 and 25 degrees C, similar to that observed with other flavodoxins. In addition, the apoflavodoxin readily binds riboflavin (Kd = 0.72 microM; 50 mM sodium phosphate, pH 7.0, 5 mM EDTA at 25 degrees C) and the complex is spectroscopically very similar to that formed with FMN. The redox potentials for the riboflavin complex were determined at pH 6.5 (E1 = -262 mV, E2 = -193 mV; 25 degrees C) and are discussed in the light of earlier proposals that charge/charge interactions between different parts of the flavin hydroquinone play a crucial role in determining E1 in flavodoxin.     

Azotobacter vinelandii flavodoxin: purification and properties of the recombinant, dephospho form expressed in Escherichia coli

The nifF gene coding for the flavodoxin from the nitrogen-fixing bacterium Azotobacter vinelandii (strain OP) was cloned into the plasmid vector pUC7 [Bennett, L. T., Jacobsen, M. R., & Dean, D. R. (1988) J. Biol. Chem. 263 1364-1369] and the resulting plasmid transformed and expressed in Escherichia coli strain DH5. Recombinant Azotobacter flavodoxin is expressed at levels 5-6-fold higher in E. coli than in comparable yields of Azotobacter cultures grown under nitrogen-fixing conditions. Even higher levels were observed with flavodoxin expressed in E. coli under control of a tac promoter. Electron spin resonance spectroscopy on whole cells and in cell-free extracts showed the flavodoxin to be largely in the semiquinone form. The flavodoxin purified from E. coli exhibited the same molecular weight, isoelectric point, flavin mononucleotide (FMN) content, N-terminal sequence, and carboxyl-terminal amino acids as for the wild-type Azotobacter protein. The recombinant flavodoxin differed from native flavodoxin in that it exhibited an increased antigenicity to flavodoxin antibody and did not contain a covalently bound phosphate. Small differences are also observed in circular dichroism spectral properties in the visible and ultraviolet spectral regions. The recombinant, dephospho flavodoxin exhibits an oxidized/semiquinone potential (pH 8.0) of -224 mV and a semiquinone/hydroquinone couple (pH 8.0) of -458 mV. This latter couple is 50-60 mV higher than that exhibited by the native flavodoxin. Resolution of recombinant dephospho flavodoxin resulted in an apoflavodoxin that was much less stable than that prepared from the native protein.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of three different flavodoxins from Azotobacter vinelandii

The flavodoxins from Azotobacter vinelandii cells grown N2-fixing and from cells grown on NH4OAc have been purified and characterized. The purified flavodoxins from these cells are a mixture of three different flavodoxins (Fld I, II, III) with different primary structures. The three proteins were separated by fast protein liquid chromatography; Fld I eluted at 0.38 M KCl, Fld II at 0.43 M KCl and Fld III at 0.45 M KCl. The most striking difference between the three flavodoxins was the midpoint potential (pH 7.0, 25 degrees C) of the semiquinone/hydroquinone couple, which was -320 mV for Fld I and -500 mV for the other two flavodoxins (Fld II and Fld III). All three flavodoxins were present in cells grown on NH4OAc. In cells grown on N2 as N source only Fld I and Fld II were found. The concentration of Fld II was 10-fold higher in N2-fixing cells than in cells grown on NH4OAc. Evidence has been obtained that Fld II is involved in electron transport to nitrogenase. As will be discussed, our observation that preparations of Azotobacter flavodoxin are heterogeneous, has consequences for the published data.     

Physicochemical properties of flavodoxin from Desulfovibrio vulgaris.

Reductive titration curves of flavodoxin from Desulfovibrio vulgaris displayed two one-electron steps. The redox potential E-2 for the couple oxidized flavodoxin/flavodoxin semiquinone was determined by direct titration with dithionite. E-2 was -149 plus or minus 3 mV (pH 7.78, 25 degrees C). The redox potential E-1 for the couple flavodoxin semiquinone/fully reduced flavodoxin was deduced from the equilibrium concentration of these species in the presence of hydrogenase and H-2. E-1 was -438 plus or minus 8 mV (pH 7.78, 25 degrees C). Light-absorption and fluorescence spectra of flavodoxin in its three redox states have been recorded. Both the rate and extent of reduction of flavodoxin semiguinone with dithionite were found to depend on pH. An equilibrium between the semiquinone and hydroquinone forms occurred at pH values close to the neutrality, even in the presence of a large excess of dithionite, suggesting an ionization in fully reduced flavodoxin with a pK-a = 6.6. The association constants K for the three FMN redox forms with the apoprotein were deduced from the value of K (K = 8 times 10-7 M-1) measured with oxidized EMN at pH 7.0. Oxidized flavodoxin was found to comproportionate with the fully reduced protein (k-comp = 4.3 times 10-3 M-1 times s-1, pH 9.0, 22 degrees C) and with reduced free FMN (K-comp = 44 M-1 times s-1, pH 8.1, 20 degrees C). Fast oxidation of reduced flavodoxin occurred in the presence of O-2. Slower oxidation of semiquinone was dependent on pH in a drastic way.     

Comparisons of wild-type and mutant flavodoxins from Anacystis nidulans. Structural determinants of the redox potentials

The long-chain flavodoxins, with 169-176 residues, display oxidation-reduction potentials at pH 7 that vary from -50 to -260 mV for the oxidized/semiquinone (ox/sq) equilibrium and are -400 mV or lower for the semiquinone/hydroquinone (sq/hq) equilibrium. To examine the effects of protein interactions and conformation changes on FMN potentials in the long-chain flavodoxin from Anacystis nidulans (Synechococcus PCC 7942), we have determined crystal structures for the semiquinone and hydroquinone forms of the wild-type protein and for the mutant Asn58Gly, and have measured redox potentials and FMN association constants. A peptide near the flavin ring, Asn58-Val59, reorients when the FMN is reduced to the semiquinone form and adopts a conformation ("O-up") in which O 58 hydrogen bonds to the flavin N(5)H; this rearrangement is analogous to changes observed in the flavodoxins from Clostridium beijerinckii and Desulfovibrio vulgaris. On further reduction to the hydroquinone state, the Asn58-Val59 peptide in crystalline wild-type A. nidulans flavodoxin rotates away from the flavin to the "O-down" position characteristic of the oxidized structure. This reversion to the conformation found in the oxidized state is unusual and has not been observed in other flavodoxins. The Asn58Gly mutation, at the site which undergoes conformation changes when FMN is reduced, was expected to stabilize the O-up conformation found in the semiquinone oxidation state. This mutation raises the ox/sq potential by 46 mV to -175 mV and lowers the sq/hq potential by 26 mV to -468 mV. In the hydroquinone form of the Asn58Gly mutant the C-O 58 remains up and hydrogen bonded to N(5)H, as in the fully reduced flavodoxins from C. beijerinckii and D. vulgaris. The redox and structural properties of A. nidulans flavodoxin and the Asn58Gly mutant confirm the importance of interactions made by N(5) or N(5)H in determining potentials, and are consistent with earlier conclusions that conformational energies contribute to the observed potentials.The mutations Asp90Asn and Asp100Asn were designed to probe the effects of electrostatic interactions on the potentials of protein-bound flavin. Replacement of acidic by neutral residues at positions 90 and 100 does not perturb the structure, but has a substantial effect on the sq/hq equilibrium. This potential is increased by 25-41 mV, showing that electrostatic interaction between acidic residues and the flavin decreases the potential for conversion of the neutral semiquinone to the anionic hydroquinone. The potentials and the effects of mutations in A. nidulans flavodoxin are rationalized using a thermodynamic scheme developed for C. beijerinckii flavodoxin.     

Preparation and properties of a cross-linked complex between ferredoxin--NADP+ reductase and flavodoxin

The electrostatically stabilized complex between Anabaena variabilis ferredoxin--NADP+ reductase and Azotobacter vinelandii flavodoxin has been covalently cross-linked by treatment with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide. The covalent complex exhibits a molecular mass and FMN/FAD content consistent with that expected for a 1:1 stoichiometry of the two flavoproteins. Immunochemical cross-reactivity is exhibited by the covalent complex with rabbit antisera prepared separately against each protein. The complex retains NADPH-ferricyanide diaphorase activity although the Km for ferricyanide is increased twofold and the turnover number is decreased by a factor of two when compared to native reductase. NADPH-cytochrome-c reductase activity of the complex is observed at a level that is quite similar to that determined at saturating concentrations of flavodoxin, while it is only 1-2% of that exhibited by the reductase in the presence of ferredoxin. No stimulation of cytochrome-c reductase activity is observed on adding ferredoxin to the cross-linked complex. Stopped-flow data show that covalent cross-linking of the flavodoxin to the reductase reduces the rate of electron transfer from its semiquinone form to cytochrome c by a factor of 60. Anaerobic titrations of the reduced complex with NADP+ show the semiquinone/quinol couple of the flavodoxin is increased 100 mV relative to the free form and the quinone/quinol couple of complexed ferredoxin-NADP+ reductase is increased by only 25 mV, relative to the free protein. Addition of NADPH to the cross-linked complex reduces the FAD of the reductase as well as the FMN moiety of flavodoxin to a mixture of semiquinone and quinol forms.     

Structure predictions and surface charge of nitrogenase flavodoxins from Klebsiella pneumoniae and Azotobacter vinelandii

A first approximation to the tertiary structure of the nitrogenase flavodoxins of Klebsiella pneumoniae and Azotobacter vinelandii can be obtained by superimposing their amino acid sequences upon the crystallographically determined structure of the long-chain flavodoxin from Anacystis nidulans. This procedure is validated by secondary structure predictions based on the sequence alone and by the distribution of polar and hydrophobic residues. It reveals, among other things, a distinctive distribution of surface charge peculiar to the nitrogenase flavodoxins, which is probably important in determining the kinetics of electron transfer with their physiological redox partners. The most likely positions of the phosphodiester bridge which has been described in the A. vinelandii molecule can also be assessed.     

Regulation of dinitrogenase reductase ADP-ribosyltransferase and dinitrogenase reductase-activating glycohydrolase by a redox-dependent conformational change of nitrogenase Fe protein

The nitrogenase-regulating enzymes dinitrogenase reductase ADP-ribosyltransferase (DRAT) and dinitrogenase reductase-activating glycohydrolase (DRAG), from Rhodospirillum rubrum, were shown to be sensitive to the redox status of the [Fe(4)S(4)](1+/2+) cluster of nitrogenase Fe protein from R. rubrum or Azotobacter vinelandii. DRAG had <2% activity with oxidized R. rubrum Fe protein relative to activity with reduced Fe protein. The activity of DRAG with oxygen-denatured Fe protein or a low molecular weight substrate, N(alpha)-dansyl-N(omega)-(1,N(6)-etheno-ADP-ribosyl)-arginine methyl ester, was independent of redox potential. The redox midpoint potential of DRAG activation of Fe protein was -430 mV versus standard hydrogen electrode, coinciding with the midpoint potential of the [Fe(4)S(4)] cluster from R. rubrum Fe protein. DRAT was found to have a specificity opposite that of DRAG, exhibiting low (<20%) activity with 87% reduced R. rubrum Fe protein relative to activity with fully oxidized Fe protein. A mutant of R. rubrum in which the rate of oxidation of Fe protein was substantially decreased had a markedly slower rate of ADP-ribosylation in vivo in response to 10 mM NH(4)Cl or darkness stimulus. It is concluded that the redox state of Fe protein plays a significant role in regulation of the activities of DRAT and DRAG in vivo.     

The vanadium- and molybdenum-containing nitrogenases of Azotobacter chroococcum. Comparison of mid-point potentials and kinetics of reduction by sodium dithionite of the iron proteins with bound magnesium adenosine 5''-diphosphate.

The mid-point potentials of the Fe protein components (Ac2 and Ac2* respectively) of the Mo nitrogenase and V nitrogenase from Azotobacter chroococcum were determined in the presence of MgADP to be -450 mV (NHE) [Ac2(MgADP)2-Ac2*ox.(MgADP)2 couple] and -463 mV (NHE) [Ac2* (MgADP)2-Ac2*ox.(ADP)2 couple] at 23 degrees C at pH 7.2. These values are consistent with a flavodoxin characterized by Deistung & Thorneley [(1986) Biochem. J. 239, 69-75] with Em = -522 mV (NHE) being an effective electron donor to both the Mo nitrogenase and the V nitrogenase in vivo. Ac2*ox.(MgADP)2 and Ac2*ox.(MgADP)2 were reduced by SO2.- (formed by the predissociation of dithionite ion, S2O4(2-)) at similar rates, k = 4.7 X 10(6) +/- 0.5 X 10(6) M-1.s-1 and 3.2 X 10(6) +/- 0.2 X 10(6) M-1.s-1 respectively, indicating structural homology at the electron-transfer site associated with the [4Fe-4S] centre in these proteins.     

Structural insight into the high reduction potentials observed for Fusobacterium nucleatum flavodoxin

Flavodoxins are small flavin mononucleotide (FMN)‐containing proteins that mediate a variety of electron transfer processes. The primary sequence of flavodoxin from Fusobacterium nucleatum, a pathogenic oral bacterium, is marked with a number of distinct features including a glycine to lysine (K13) substitution in the highly conserved phosphate‐binding loop (T/S‐X‐T‐G‐X‐T), variation in the aromatic residues that sandwich the FMN cofactor, and a more even distribution of acidic and basic residues. The E_ox/sq (oxidized/semiquinone; ?43 mV) and E_sq/hq (semiquinone/hydroquinone; ?256 mV) are the highest recorded reduction potentials of known long‐chain flavodoxins. These more electropositive values are a consequence of the apoprotein binding to the FMN hydroquinone anion with ~70‐fold greater affinity compared to the oxidized form of the cofactor. Inspection of the FnFld crystal structure revealed the absence of a hydrogen bond between the protein and the oxidized FMN N5 atom, which likely accounts for the more electropositive E_ox/sq. The more electropositive E_sq/hq is likely attributed to only one negatively charged group positioned within 12 Å of the FMN N1. We show that natural substitutions of highly conserved residues partially account for these more electropositive reduction potentials.     

Cloning, sequencing and expression of the gene for flavodoxin from Megasphaera elsdenii and the effects of removing the protein negative charge that is closest to N(1) of the bound FMN.

The gene for the electron-transfer protein flavodoxin has been cloned from Megasphaera elsdenii using the polymerase chain reaction. The recombinant gene was sequenced, expressed in an Escherichia coli expression system, and the recombinant protein purified and characterized. With the exception of an additional methionine residue at the N-terminus, the physico-chemical properties of the protein, including its optical spectrum and oxidation-reduction properties, are very similar to those of native flavodoxin. A site-directed mutant, E60Q, was made to investigate the effects of removing the negatively charged group that is nearest to N(1) of the bound FMN. The absorbance maximum in the visible region of the bound flavin moves from 446 to 453 nm. The midpoint oxidation-reduction potential at pH 7 for reduction of oxidized flavodoxin to the semiquinone E2 becomes more negative, decreasing from -114 to -242 mV; E1, the potential for reduction of semiquinone to the hydroquinone, becomes less negative, increasing from -373 mV to -271 mV. A redox-linked pKa associated with the hydroquinone is decreased from 5.8 to < or = 4.3. The spectra of the hydroquinones of wild-type and mutant proteins depend on pH (apparent pKa values of 5.8 and < or = 5.2, respectively). The complexes of apoprotein and all three redox forms of FMN are much weaker for the mutant, with the greatest effect occurring when the flavin is in the semiquinone form. These results suggest that glutamate 60 plays a major role in control of the redox properties of M. elsdenii flavodoxin, and they provide experimental support to an earlier proposal that the carboxylate on its side-chain is associated with the redox-linked pKa of 5.8 in the hydroquinone.     

Role of glutamate-59 hydrogen bonded to N(3)H of the flavin mononucleotide cofactor in the modulation of the redox potentials of the Clostridium beijerinckii flavodoxin. Glutamate-59 is not responsible for the pH dependency but contributes to the stabilization of the flavin semiquinone.

The midpoint potentials for both redox couples of the noncovalently bound flavin mononucleotide (FMN) cofactor in the flavodoxin are known to be pH dependent. While the pH dependency for the oxidized-semiquinone (ox/sq) couple is consistent with the formation of the blue neutral form of the flavin semiquinone, that of the semiquinone-hydroquinone (sq/hq) couple is more enigmatic. The apparent pK(a) of 6.7 for this couple in the flavodoxin from Clostridium beijerinckii has been attributed to the ionization of the FMN(HQ); however, nuclear magnetic resonance data strongly suggest the FMN(HQ) remains anionic over the entire pH range testable. As an alternative explanation, a specific glutamate residue (Glu59 in this flavodoxin), which is hydrogen-bonded to N(3)H of the FMN, has been postulated to be the primary redox-linked proton acceptor responsible for the pH effect in some flavodoxins. This model was directly tested in this study by permanently neutralizing Glu59 by its replacement with glutamine. This conservative substitution resulted in an increase of 86 mV (at pH 7) in midpoint potential of the sq/hq couple; however, the pH dependency of this couple was not altered. Thus, the redox-linked protonation of Glu59 clearly cannot be responsible for this effect as proposed. The pH dependency of the ox/sq couple was also similar to wild type, but the midpoint potential has decreased by 65 mV (pH 7). The K(d) values for the oxidized, semiquinone, and hydroquinone complexes increased by 43-, 590-, and 20-fold, respectively, relative to the wild type. Thus, the Glu59 to glutamine substitution substantially effects the stability of the semiquinone but, on a relative basis, slightly favors the formation of the hydroquinone. On the basis of (1)H-(15)N HSQC nuclear magnetic resonance spectroscopic studies, the increased temperature coefficients for the protons on N(3) and N(5) of the reduced FMN in E59Q suggest that the hydrogen-bonding interactions at these positions are significantly weakened in this mutant. The increase for N(5)H correlates with the reduced stability of the FMN(SQ) and the more negative midpoint potential for the ox/sq couple. On the basis of the X-ray structure, an "anchoring" role is proposed for the side chain carboxylate of Glu59 that stabilizes the structure of the 50's loop in such a way so as to promote the crucial hydrogen-bonding interaction that stabilizes the flavin semiquinone, contributing to the low potential of this flavodoxin.     

Further characterisation of the FAD and Fe2S2 redox centres of component C, the NADH:acceptor reductase of the soluble methane monooxygenase of Methylococcus capsulatus (Bath)

The absorbance contributions of the FAD and Fe2S2 redox centres of component C of the soluble methane monooxygenase complex have been resolved, using mersalyl to destroy the Fe2S2 centre. The Fe2S2 seems to be very similar to that of spinach ferredoxin, by its absorbance and electron paramagnetic resonance (EPR) spectra, and the FAD semiquinone is a neutral semiquinone. Spectrophotometry near room temperature and EPR spectroscopy near liquid-helium temperature allow the three redox couples of component C to be ordered. Component C can exist in Oe-1 (oxidised), 1e-1 (semiquinone), 2e-1 (mostly semiquinone and reduced Fe2S2), and 3e-1 forms (dihydroquinone and reduced Fe2S2), under equilibrium conditions. The ability of component C to support odd-electron forms is consistent with its proposed role as a 2e-1/1e-1 transformase, splitting electron pairs from NADH for passage to component A in one-electron steps. (The FAD appears to interact with NADH, and transfers single electrons to the Fe2S2, for donation to component A at a constant redox potential.) The mid-point potentials of component C were found using redox dyes and EPR spectroscopy and were: FAD/FAD., Em = -150 mV; Fe2S2/Fe2.S2,Em = -220 mV; FAD./FAD..,Em = -260 mV. the presence of NADH did not alter these mid-point potentials. These mid-point potentials are consistent with the role of component C as the NADH:component A reductase, passing electrons from NADH (Em = -320 mV) onto component A (Em = +150 mV and Em = -150 mV). The reducing power from NADH appears to be required by component A to activate one atom of oxygen, to insert into methane, and the reducing equivalents derived from NADH end up with the other oxygen atom, as water.     

Factors affecting redox potential and differential sensitivity of SoxR to redox‐active compounds

SoxR is a [2Fe‐2S]‐containing sensor‐regulator, which is activated through oxidation by redox‐active compounds (RACs). SoxRs show differential sensitivity to RACs, partly due to different redox potentials, such that Escherichia coli (Ec) SoxR with lower potential respond to broader range of RACs than Streptomyces coelicolor (Sc) SoxR. In S. coelicolor, the RACs that do not activate ScSoxR did not inhibit growth, suggesting that ScSoxR is tuned to respond to growth‐inhibitory RACs. Based on sequence comparison and mutation studies, two critical amino acids around the [2Fe‐2S] binding site were proposed as key determinants of sensitivity. ScSoxR‐like mutation (R127L/P131V) in EcSoxR changed its sensitivity profile as ScSoxR, whereas EcSoxR‐like mutation (L126R/V130P) in ScSoxR caused relaxed response. In accordance, the redox potentials of EcSoxR^R127L/P131V and ScSoxR^L126R/V130P were estimated to be ?192 ± 8 mV and ?273 ± 10 mV, respectively, approaching that of ScSoxR (?185 mV) and EcSoxR (?290 mV). Molecular dynamics simulations revealed that the R127L and P131V substitutions in EcSoxR caused more electropositive environment around [2Fe‐2S], making it harder to get oxidized. This reveals a mechanism to modulate redox‐potential in [Fe‐S]‐containing sensors by point mutations and to evolve a sensor with differential sensitivity to achieve optimal cellular physiology.     

Electron transfer to nitrogenase. Characterization of flavodoxin from Azotobacter chroococcum and comparison of its redox potentials with those of flavodoxins from Azotobacter vinelandii and Klebsiella pneumoniae (nifF-gene product).

Flavodoxin in the hydroquinone state acts as an electron donor to nitrogenase in several nitrogen-fixing organisms. The mid-point potentials for the oxidized-semiquinone and semiquinone-hydroquinone couples of flavodoxins isolated from facultative anaerobe Klebsiella pneumoniae (nifF-gene product, KpFld) and the obligate aerobe Azotobacter chroococcum (AcFld) were determined as a function of pH. The mid-point potentials of the semiquinone-hydroquinone couples of KpFld and AcFld are essentially independent of pH over the range pH 7-9, being -422 mV and -522 mV (normal hydrogen electrode) at pH 7.5 respectively. The mid-point potentials of the quinone-semiquinone couples at pH 7.5 are -200 mV (KpFld) and -133 mV (AcFld) with delta Em/pH of -65 +/- 4 mV (KpFld) and -55 +/- 2 mV (AcFld) over the range pH 7.0-9.5. This indicates that reduction of the quinone is coupled to protonation to yield a neutral semiquinone. The significance of these values with respect to electron transport to nitrogenase is discussed. The amino acid compositions, the N- and C-terminal amino acid sequences and the u.v.-visible spectra of KpFld and AcFld were determined and are compared with published data for flavodoxins isolated from Azotobacter vinelandii.     

The redox properties of ascorbate peroxidase

Reduction potentials for the catalytic compound I/compound II and compound II/Fe3+ redox couples, and for the two-electron compound I/Fe3+ redox couple, have been determined for ascorbate peroxidase (APX) and for a number of site-directed variants. For the wild type enzyme, the values are E degrees '(compound I/compound II) = 1156 mV, E degrees '(compound II/Fe3+) = 752 mV, and E degrees '(compound I/Fe3+) = 954 mV. For the variants, the analysis also includes determination of Fe3+/Fe2+ potentials which were used to calculate (experimentally inaccessible) E degrees '(compound II/Fe3+) potentials. The data provide a number of new insights into APX catalysis. The measured values for E degrees '(compound I/compound II) and E degrees '(compound II/Fe3+) for the wild type protein account for the much higher oxidative reactivity of compound I compared to compound II, and this correlation holds for a number of other active site and substrate binding variants of APX. The high reduction potential for compound I also accounts for the known thermodynamic instability of this intermediate, and it is proposed that this instability can account for the deviations from standard Michaelis kinetics observed for most APX enzymes during steady-state oxidation of ascorbate. This study provides the first systematic evaluation of the redox properties of any ascorbate peroxidase using a number of methods, and the data provide an experimental and theoretical framework for accurate determination of the redox properties of Fe3+, compound I, and compound II species in related enzymes.     

A crystallographic study of Cys69Ala flavodoxin II from Azotobacter vinelandii: structural determinants of redox potential

Flavodoxin II from Azotobacter vinelandii is a "long-chain" flavodoxin and has one of the lowest E1 midpoint potentials found within the flavodoxin family. To better understand the relationship between structural features and redox potentials, the oxidized form of the C69A mutant of this flavodoxin was crystallized and its three-dimensional structure determined to a resolution of 2.25 A by molecular replacement. Its overall fold is similar to that of other flavodoxins, with a central five-stranded parallel beta-sheet flanked on either side by alpha-helices. An eight-residue insertion, compared with other long-chain flavodoxins, forms a short 3(10) helix preceding the start of the alpha3 helix. The flavin mononucleotide (FMN) cofactor is flanked by a leucine on its re face instead of the more conserved tryptophan, resulting in a more solvent-accessible FMN binding site and stabilization of the hydroquinone (hq) state. In particular the absence of a hydrogen bond to the N5 atom of the oxidized FMN was identified, which destabilizes the ox form, as well as an exceptionally large patch of acidic residues in the vicinity of the FMN N1 atom, which destabilizes the hq form. It is also argued that the presence of a Gly at position 58 in the sequence stabilizes the semiquinone (sq) form, as a result, raising the E2 value in particular.     

Molecular dissection of human methionine synthase reductase: determination of the flavin redox potentials in full-length enzyme and isolated flavin-binding domains

Human methionine synthase reductase (MSR) catalyzes the NADPH-dependent reductive methylation of methionine synthase. MSR is 78 kDa flavoprotein belonging to a family of diflavin reductases, with cytochrome P450 reductase (CPR) as the prototype. MSR and its individual flavin-binding domains were cloned as GST-tagged fusion proteins for expression and purification from Escherichia coli. The isolated flavin domains of MSR retain UV-visible and secondary structural properties indicative of correctly folded flavoproteins. Anaerobic redox titrations on the individual domains assisted in assignment of the midpoint potentials for the high- and low-potential flavin. For the isolated FMN domain, the midpoint potentials for the oxidized/semiquinone (ox/sq) couple and semiquinone/hydroquinone (sq/hq) couple are -112 and -221 mV, respectively, at pH 7.0 and 25 degrees C. The corresponding couples in the isolated FAD domain are -222 mV (ox/sq) and -288 mV (sq/hq). Both flavins form blue neutral semiquinone species characterized by broad absorption peaks in the long-wavelength region during anaerobic titration with sodium dithionite. In full-length MSR, the values of the FMN couples are -109 mV (ox/sq) and -227 mV (sq/hq), and the corresponding couple values for FAD are -254 mV (ox/sq) and -291 mV (sq/hq). Separation of the MSR flavins does not perturb their thermodynamic properties, as midpoint potentials for all four couples are similar in isolated domains and in full-length MSR. The redox properties of MSR are discussed in relation to other members of the diflavin oxidoreductase family and the mechanism of electron transfer.