Flavodoxin Semiquinone Research Articles

Spectroscopic, thermodynamic and computational evidence of the locations of the FADs in the nitrogen fixation-associated electron transfer flavoprotein.

Flavin-based electron bifurcation allows enzymes to redistribute energy among electrons by coupling endergonic and exergonic electron transfer reactions. Diverse bifurcating enzymes employ a two-flavin electron transfer flavoprotein (ETF) that accepts hydride from NADH at a flavin (the so-called bifurcating FAD, Bf-FAD). The Bf-FAD passes one electron exergonically to a second flavin thereby assuming a reactive semiquinone state able to reduce ferredoxin or flavodoxin semiquinone. The flavin that accepts one electron and passes it on via exergonic electron transfer is known as the electron transfer FAD (ET-FAD) and is believed to correspond to the single FAD present in canonical ETFs, in domain II. The Bf-FAD is believed to be the one that is unique to bifurcating ETFs, bound between domains I and III. This very reasonable model has yet to be challenged experimentally. Herein we used site-directed mutagenesis to disrupt FAD binding to the presumed Bf site between domains I and III, in the Bf-ETF from Rhodopseudomonas palustris (RpaETF). The resulting protein contained only 0.80 ± 0.05 FAD, plus 1.21 ± 0.04 bound AMP as in canonical ETFs. The flavin was not subject to reduction by NADH, confirming absence of Bf-FAD. The retained FAD displayed visible circular dichroism (CD) similar to that of the ET-FAD of RpaETF. Likewise, the mutant underwent two sequential one-electron reductions forming and then consuming anionic semiquinone, reproducing the reactivity of the ET-FAD. These data confirm that the retained FAD in domain II corresponds the ET-FAD. Quantum chemical calculations of the absorbance and CD spectra of each of WT RpaETF's two flavins reproduced the observed differences between their CD and absorbance signatures. The calculations for the flavin bound in domain II agreed better with the spectra of the ET-flavin, and those calculated based on the flavin between domains I and III agreed better with spectra of the Bf-flavin. Thus calculations independently confirm the locations of each flavin. We conclude that the site in domain II harbours the ET-FAD whereas the mutated site between domains I and III is the Bf-FAD site, confirming the accepted model by two different tests.

Reduction midpoint potentials of bifurcating electron transfer flavoproteins.

Recently, a variety of enzymes have been found to accept electrons from NAD(P)H yet reduce lower-potential carriers such as ferredoxin and flavodoxin semiquinone, in apparent violation of thermodynamics. The reaction is favorable overall, however, because these enzymes couple the foregoing endergonic one-electron transfer to exergonic transfer of the other electron from each NAD(P)H, in a process called "flavin-based electron bifurcation." The reduction midpoint potentials (E°s) of the multiple flavins in these enzymes are critical to their mechanisms. We describe methods we have found to be useful for measuring each of the E°s of each of the flavins in bifurcating electron transfer flavoproteins.

The Electron Bifurcating FixABCX Protein Complex from Azotobacter vinelandii: Generation of Low-Potential Reducing Equivalents for Nitrogenase Catalysis.

The biological reduction of dinitrogen (N2) to ammonia (NH3) by nitrogenase is an energetically demanding reaction that requires low-potential electrons and ATP; however, pathways used to deliver the electrons from central metabolism to the reductants of nitrogenase, ferredoxin or flavodoxin, remain unknown for many diazotrophic microbes. The FixABCX protein complex has been proposed to reduce flavodoxin or ferredoxin using NADH as the electron donor in a process known as electron bifurcation. Herein, the FixABCX complex from Azotobacter vinelandii was purified and demonstrated to catalyze an electron bifurcation reaction: oxidation of NADH (Em = -320 mV) coupled to reduction of flavodoxin semiquinone (Em = -460 mV) and reduction of coenzyme Q (Em = 10 mV). Knocking out fix genes rendered Δrnf A. vinelandii cells unable to fix dinitrogen, confirming that the FixABCX system provides another route for delivery of electrons to nitrogenase. Characterization of the purified FixABCX complex revealed the presence of flavin and iron-sulfur cofactors confirmed by native mass spectrometry, electron paramagnetic resonance spectroscopy, and transient absorption spectroscopy. Transient absorption spectroscopy further established the presence of a short-lived flavin semiquinone radical, suggesting that a thermodynamically unstable flavin semiquinone may participate as an intermediate in the transfer of an electron to flavodoxin. A structural model of FixABCX, generated using chemical cross-linking in conjunction with homology modeling, revealed plausible electron transfer pathways to both high- and low-potential acceptors. Overall, this study informs a mechanism for electron bifurcation, offering insight into a unique method for delivery of low-potential electrons required for energy-intensive biochemical conversions.

The Ferredoxin-NADP(H) Reductase fromRhodobacter capsulatus: Molecular Structure and Catalytic Mechanism,

The photosynthetic bacterium Rhodobacter capsulatus contains a ferredoxin (flavodoxin)-NADP(H) oxidoreductase (FPR) that catalyzes electron transfer between NADP(H) and ferredoxin or flavodoxin. The structure of the enzyme, determined by X-ray crystallography, contains two domains harboring the FAD and NADP(H) binding sites, as is typical of the FPR structural family. The FAD molecule is in a hairpin conformation in which stacking interactions can be established between the dimethylisoalloxazine and adenine moieties. The midpoint redox potentials of the various transitions undergone by R. capsulatus FPR were similar to those reported for their counterparts involved in oxygenic photosynthesis, but its catalytic activity is orders of magnitude lower (1-2 s(-)(1) versus 200-500 s(-)(1)) as is true for most of its prokaryotic homologues. To identify the mechanistic basis for the slow turnover in the bacterial enzymes, we dissected the R. capsulatus FPR reaction into hydride transfer and electron transfer steps, and determined their rates using stopped-flow methods. Hydride exchange between the enzyme and NADP(H) occurred at 30-150 s(-)(1), indicating that this half-reaction does not limit FPR activity. In contrast, electron transfer to flavodoxin proceeds at 2.7 s(-)(1), in the range of steady-state catalysis. Flavodoxin semiquinone was a better electron acceptor for FPR than oxidized flavodoxin under both single turnover and steady-state conditions. The results indicate that one-electron reduction of oxidized flavodoxin limits the enzyme activity in vitro, and support the notion that flavodoxin oscillates between the semiquinone and fully reduced states when FPR operates in vivo.

Lys35 of PsaC is required for the efficient photoreduction of flavodoxin by photosystem I from Chlamydomonas reinhardtii.

The photoreduction of the oxidized and the semiquinone form of flavodoxin from Synechocystis sp. PCC 6803 by the photosystem I (PSI) of wild-type Chlamydomonas reinhardtii and the mutant strains Lys35Asp, Lys35Glu and Lys35Arg was analysed by flash-absorption spectroscopy to investigate the role of residue Lys35 of the PSI subunit PsaC in flavodoxin reduction. For PSI preparations from C. reinhardtii the reduction of oxidized flavodoxin was monoexponential and approached limiting electron transfer rates similar to those of cyanobacterial PSI from the wild-type and the Lys35Arg mutant. For PSI from the Lys35Glu mutant, however, a approximately 2.5-fold smaller value was determined. The photoreduction of flavodoxin semiquinone by PSI from C. reinhardtii lacked fast first-order kinetic components and, in contrast with PSI from cyanobacteria, displayed only a single concentration-dependent phase. From this phase, second-order rate constants were calculated for wild-type PSI and PSI from the Lys35Arg mutant which were comparable to those of PSI from cyanobacteria. For PSI from the Lys35Glu and the Lys35Asp mutants the derived second-order rate constants were 19 and 10 times smaller. Thus, the inversion of charge at position 35 of PsaC negatively affects the rate of electron transfer to both forms of flavodoxin, whereas PSI complexes that retain a positive charge at this position show wild-type kinetics. However, the positive charge at this position of PsaC is not essential for flavodoxin photoreduction as the number of flavodoxin molecules reduced per PSI was similar for all of the PSI complexes investigated. In addition, chemical cross-linking assays showed that the binary cross-linking product between flavodoxin and PsaC of PSI from wild-type C. reinhardtii was not formed with PSI complexes from the Lys13Asp and Lys35Glu mutants. This indicates that Lys35 of PsaC is probably essential for the chemical cross-link between PsaC and flavodoxin. Taken together, these experiments show that Lys35 of PsaC plays a strikingly similar role in the electron transfer from PSI to both ferredoxin and flavodoxin.

Laser-flash absorption spectroscopy study of the competition between ferredoxin and flavodoxin photoreduction by Photosystem I in Synechococcus sp. PCC 7002: Evidence for a strong preference for ferredoxin

The competition between ferredoxin and flavodoxin for electrons from Photosystem I was analyzed by flash absorption spectroscopy of the photoreduction processes that take place in the presence of both acceptor proteins in vitro. Steady state photoreduction assays indicate a strong inhibition of the apparent flavodoxin photoreduction activities of Photosystem I in the presence of ferredoxin. Flash-absorption experiments carried out at 626 nm, a wavelength where the reduction of ferredoxin shows no spectral contribution, show that the photoreduction of oxidized flavodoxin and flavodoxin semiquinone are inhibited by ferredoxin in a quantitatively similar way. The experimental data can be satisfactorily described by a reaction model that assumes that both redox states of flavodoxin do not compete with ferredoxin for binding on PS I and that the binding equilibrium between ferredoxin and PS I is not changed in their presence. In contrast, a model which assumes that ferredoxin and flavodoxin actually compete for binding to PS I gives poor results. Similarly, experimental data observed in the presence of both redox states of flavodoxin can also be quantitatively described under the assumption that the binding equilibrium between flavodoxin semiquinone and PS I is not disturbed by oxidized flavodoxin. Taken together, this analysis shows that PS I favors ferredoxin over flavodoxin and flavodoxin semiquinone over oxidized flavodoxin. This behavior is in accordance with the values of the dissociation constants for complexes between PS I and its acceptors. However, in case of ferredoxin the observed preference is stronger than expected from these values, indicating that ferredoxin is almost absolutely preferred by PS I over flavodoxin and is always reduced first.

Characterisation of flavodoxin NADP+ oxidoreductase and flavodoxin; key components of electron transfer in Escherichia coli.

The genes encoding the Escherichia coli flavodoxin NADP+ oxidoreductase (FLDR) and flavodoxin (FLD) have been overexpressed in E. coli as the major cell proteins (at least 13.5% and 11.4% of total soluble protein, respectively) and the gene products purified to homogeneity. The FLDR reduces potassium ferricyanide with a kcat of 1610.3 min(-1) and a Km of 23.6 microM, and cytochrome c with a kcat of 141.3 min(-1) and a Km of 17.6 microM. The cytochrome c reductase rate is increased sixfold by addition of FLD and an apparent Km of 6.84 microM was measured for the affinity of the two flavoproteins. The molecular masses of FLDR and FLD apoproteins were determined as 27648 Da and 19606 Da and the isoelectric points as 4.8 and 3.5, respectively. The mass of the FLDR is precisely that predicted from the atomic structure and indicates that residue 126 is arginine, not glutamine as predicted from the gene sequence. FLDR and FLD were covalently crosslinked using 1-ethyl-3(dimethylamino-propyl) carbodiimide to generate a catalytically active heterodimer. The midpoint reduction potentials of the oxidised/semiquinone and semiquinone/hydroquinone couples of both FLDR (-308 mV and -268 mV, respectively) and FLD (-254 mV and -433 mV, respectively) were measured using redox potentiometry. This confirms the electron-transfer route as NADPH-->FLDR-->FLD. Binding of 2' adenosine monophosphate increases the midpoint reduction potentials for both FLDR couples. These data highlight the strong stabilisation of the flavodoxin semiquinone (absorption coefficient calculated as 4933 M(-1) cm(-1) at 583 nm) with respect to the hydroquinone state and indicate that FLD must act as a single electron shuttle from the semiquinone form in its support of cellular functions, and to facilitate catalytic activity of microsomal cytochromes P-450 heterologously expressed in E. coli. Kinetic studies of electron transfer from FLDR/FLD to the fatty acid oxidase P-450 BM3 support this conclusion, indicating a ping-pong mechanism. This is the first report of the potentiometric analysis of the full E. coli NAD(P)H/FLDR/FLD electron-transfer chain; a complex critical to the function of a large number of E. coli redox systems.

The PsaE subunit is required for complex formation between photosystem I and flavodoxin from the cyanobacterium Synechocystis sp. PCC 6803.

The photoreduction of the oxidized and the semiquinone form of flavodoxin by photosystem I particles (PSI) from the wild type and a psaE deletion strain from the cyanobacterium Synechocystis sp. PCC 6803 was analyzed by flash-absorption spectroscopy to investigate a possible involvement of the PsaE subunit in this photoreduction process. The kinetics of the reduction of oxidized flavodoxin display a single-exponential component for both PSI preparations. Limiting electron transfer rates kobs of approximately 500 and approximately 900 s -1 are deduced for the wild type and PSI from the psaE-less mutant, respectively, indicating that the PsaE subunit is not important for this photoreduction process. In the case of wild-type PSI, the reduction of flavodoxin semiquinone is a biphasic process, displaying a fast first-order phase with a t1/2 of approximately 13 micro(s) which is then followed by a slower, concentration-dependent phase, for which a second-order rate constant k2 of 2.2 x 10(8) M-1 cm-1 is calculated. In contrast, photoreduction of the semiquinone by PSI from the psaE-less mutant is monoexponential, displaying only one second-order component with a second-order rate constant similar to those observed for wild-type PSI (k2 = 1.5 x 10(8) M-1 cm-1). The fast first-order component which is interpreted as an electron transfer process within a preformed complex between flavodoxin semiquinone and PSI is almost completely absent in the reduction of flavodoxin by the PsaE-less PSI. A similar loss of the fast phase is also observed for the photoreduction of flavodoxin semiquinone by PSI from a Synechococcus elongatus psaE-less mutant. Upon reconstitution of isolated PsaE to the PsaE-less PSI in vitro, approximately 80% of the fast first-order kinetic component is recovered, indicating that PsaE is required for high-affinity binding of the flavodoxin semiquinone to PSI. In addition, chemical cross-linking assays show that flavodoxin can no longer be cross-linked to PSI in detectable amounts when PsaE is missing on the reaction center. Taken together, these experiments indicate that the PsaE subunit is required for complex formation between PSI and flavodoxin but is not required for an efficient forward electron transfer from photosystem I to both forms of flavodoxin.

Characterization of a redox-active cross-linked complex between cyanobacterial photosystem I and its physiological acceptor flavodoxin.

A covalent complex between photosystem I and flavodoxin from the cyanobacterium Synechococcus sp. PCC 7002 was generated by chemical cross-linking. Laser flash-absorption spectroscopy indicates that the bound flavodoxin of this complex is stabilized in the semiquinone state and is photoreduced to the quinol form upon light excitation. The kinetics of this photoreduction process, which takes place in approximately 50% of the reaction centres, displays three exponential components with half-lives of 9 microsec, 70 microsec and 1 ms. The fully reduced flavodoxin subsequently recombines with P700+ with a t1/2 of 330 ms. A corresponding flavodoxin semiquinone radical signal is readily observed in the dark by room temperature electron paramagnetic resonance, which reversibly disappears upon illumination. In contrast, the light-induced reduction of oxidized flavodoxin can be observed only by first-flash experiments following excessive dark adaptation. In addition, the docking site of flavodoxin on photosystem I was determined by electron microscopy in combination with image analysis. Flavodoxin binds to the cytoplasmic side of photosystem I at a distance of 7 nm from the centre of the trimer and in close contact to a ridge formed by the subunits PsaC, PsaD and PsaE.

Laser flash absorption spectroscopy study of flavodoxin reduction by photosystem I in Synechococcus sp. PCC 7002.

The photoreduction of flavodoxin by trimeric photosystem I, both from the cyanobacterium Synechococcus sp. PCC 7002, was investigated by flash absorption spectroscopy. After addition of flavodoxin in darkness, single flash experiments show that the transient signals change between individual flashes. This behavior is assigned to a progressive accumulation of flavodoxin semiquinone, which is relatively stable under most experimental conditions. Different conditions were devised in order to study the reduction of the oxidized and semiquinone forms of flavodoxin separately. Both processes were identified by their differential spectra measured between 460 and 630 nm. Detailed kinetic characteristics of flavodoxin reduction were obtained at pH 8.0 in the presence of salts. The kinetics of reduction of oxidized flavodoxin displays a single-exponential component. The rate of this component increases with the flavodoxin concentration up to an asymptotic value of about 600 s-1. The semiquinone form of flavodoxin being protonated, this rate corresponds to a rate-limiting reaction which could be either an electron transfer reaction or a protonation reaction. In contrast, the reduction of flavodoxin semiquinone is biphasic. A fast first-order phase with t 1/2 approximately 10 microseconds is interpreted as an electron transfer process within a preformed complex. A dissociation constant of 2.64 microM is calculated for this complex by assuming a simple binding equilibrium between photosystem I and flavodoxin semiquinone. The slower phase observed for semiquinone reduction is concentration dependent, and a second-order rate constant of 1.7 x 10(8) M-1 s-1 is calculated. For both one-electron reduction steps, different optimal salt concentrations are observed indicating slightly different interactions between photosystem I and flavodoxin in its oxidized and semiquinone states.

Comparison of Electron Transfer Kinetics between Redox Proteins Free in Solution and Electrostatically Complexed to a Lipid Bilayer Membrane

The second-order rate constants obtained in solution for the reduction of horse cytochrome c (cytc; net charge +7) by either Clostridium beijerinckii flavodoxin semiquinone (Fld; net charge −16) or reduced spinach ferredoxin (Fd; net charge −15) decrease monotonically with increasing ionic strength, as expected for reactions between oppositely charged species. Although the rate constant for the Fld reaction is almost two orders of magnitude larger at low ionic strength than that for Fd, the values extrapolated to infinite ionic strength are closely similar, indicating comparable reactivities when electrostatic effects are eliminated. Furthermore, Fld has a much larger value for the electrostatic interaction energy, and thus a larger apparent active site charge, than does Fd, accounting for the rate constant disparity at low ionic strength. Electrostatically binding cytc at low ionic strength to a negatively charged Lipid bilayer vesicle (membranes containing mixtures of egg phosphatidylcholine (PC) and cardiolipin (CL)) results in a marked decrease of the observed electron transfer rate constant ( k obs) for reduction of the cytochrome by both Fld and Fd. The magnitude of this decrease is proportional to the mole percent of CL present in the membrane (10- to 20-fold change over 5-60 mol%). With Fld, k obs decreases monotonically with increasing ionic strength at a fixed CL concentration. With Fd an increase in k obs occurs as the ionic strength is increased, which maximizes at intermediate ionic strength at a value larger than that obtained in the absence of lipid vesicles. When Fld is electrostatically bound to a positively charged vesicle composed of 40 mol% dioctadecyldimethylammonium ion (DODAC) and 60 mol% PC, again k obs for electron transfer to cytc is decreased over that obtained in solution, and the magnitude is diminished monotonically by increasing ionic strength. In contrast, k obs for electron transfer from Fd to cytc is unaffected by the presence of the positively charged membrane. The implications of these results for the role of membrane surface charge in modulating protein-protein interactions is discussed.

A 31P‐nuclear‐magnetic‐resonance study of NADPH–cytochrome‐P‐450 reductase and of the Azotobacter flavodoxin/ferredoxin‐NADP+ reductase complex

31P-nuclear-magnetic-resonance spectroscopy has been employed to probe the structure of the detergent-solubilized form of liver microsomal NADPH--cytochrome-P-450 reductase. In addition to the resonances due to the FMN and FAD coenzymes, additional phosphorus resonances are observed and are assigned to the tightly bound adenosine 2'-phosphate (2'-AMP) and to phospholipids. The phospholipid content was found to vary with the preparation; however, the 2'-AMP resonance was observed in all preparations tested. In agreement with published results [Otvos et al. (1986) Biochemistry 25, 7220-7228] for the protease-solubilized enzyme, the addition of Mn(II) to the oxidized enzyme did not result in any observable line-broadening of the FMN and FAD phosphorus resonances. The phospholipid resonances, however, were extensively broadened and the line width of the phosphorus resonance assigned to the bound 2'-AMP was broadened by approximately 70 Hz. The data show that only the phosphorus moieties of the phospholipids and the 2'-AMP, but not the flavin coenzymes are exposed to the bulk solvent. Removal of the FMN moiety from the enzyme substantially alters the 31P-NMR spectrum as compared with the native enzyme. The 2'-AMP is removed from the enzyme during the FMN-depletion procedure and the pyrophosphate resonances of the bound FAD are significantly altered. Reconstitution of the FMN-depleted protein with FMN results in the restoration of the coenzyme spectral properties. Reduction of FMN to its air-stable paramagnetic semiquinone form results in broadening of the FMN and 2'-AMP resonances in the detergent-solubilized enzyme. In agreement with previous results. FMN semiquinone formation had little or no effect on the line width of the FMN phosphorus resonance for the proteolytically solubilized enzyme. 31P-NMR experiments with Azotobacter flavodoxin semiquinone, both in its free form and in a complex with spinach ferredoxin-NADP+ reductase, mimic the differential paramagnetic effects of the flavin semiquinone on the line width of the FMN phosphorus resonance, observed by comparison of the detergent-solubilized and protease-solubilized forms of the reductase. The data demonstrate that assignment of the site of flavin semiquinone formation to a particular flavin coenzyme may not always be possible by 31P-NMR experiments in multi-flavin containing enzymes.

Electrostatic effects on the spectral and redox properties of Clostridium pasteurianum flavodoxin: Effects of salt concentration and polylysine

When polylysine is complexed to flavodoxin at low ionic strength, the electrostatic potential of the region which is involved in electron transfer is modified such that positively charged oxidants react more slowly with flavodoxin semiquinone, and negatively charged oxidants react more rapidly. The reaction rate of the uncharged benzoquinone molecule is unaffected. An especially strong effect (approximately 200-fold) occurs with ferricyanide. This is interpreted in terms of electrostatic control of the reaction site. Complexation also changes the conformation of the region around the FMN prosthetic group, which is reflected in the fluorescence and circular dichroism spectra of the protein.

Transient kinetics of reduction of blue copper proteins by free flavin and flavodoxin semiquinones.

Rate constants have been determined for the electron-transfer reactions between reduced free flavins and flavodoxin semiquinone and several blue copper proteins. Correlations between these values and redox potentials demonstrate that spinach plastocyanin, Pseudomonas aeruginosa azurin, Alcaligenes sp. azurin, and Alcaligenes sp. nitrite reductase have the same intrinsic reactivities toward free flavins, whereas stellacyanin is more reactive (3.3 times) and laccase considerably less reactive (approximately 12 times). Electrostatic interactions between the negatively charged flavin mononucleotide (FMN) and the copper proteins show that the interaction site charges for laccase and nitrite reductase are opposite in sign to the net protein charge and that the signs and magnitudes of the charges are consistent with the known three-dimensional structures for plastocyanin and the azurins and with amino acid sequence homologies for stellacyanin. The results demonstrate that the apparent interaction site charge with flavodoxin is larger than that with FMN for plastocyanin, nitrite reductase, and stellacyanin but smaller for Pseudomonas azurin. This is interpreted in terms of a larger interaction domain for the flavodoxin reaction, which allows charged groups more distant from the actual electron-transfer site to become involved. The intrinsic reactivities of plastocyanin and azurin toward flavodoxin are the same, as was the case with FMN, but both stellacyanin and nitrite reductase are considerably less reactive than expected (approximately 2 orders of magnitude). This result suggests the involvement of steric factors with these latter two proteins which discriminate against large reactants such as flavodoxin.

Kinetics of electron transfer between cytochromes c' and the semiquinones of free flavin and clostridial flavodoxin.

Rate constants have been measured for the reactions of a series of high-spin cytochromes c' and their low-spin homologues (cytochromes c-554 and c-556) with the semiquinones of free flavins and flavodoxin. These cytochromes are approximately 3 times more reactive with lumiflavin and riboflavin semiquinones than are the c-type cytochromes that are homologous to mitochondrial cytochrome c. We attribute this to the greater solvent exposure of the heme in the c'-type cytochromes. In marked contrast, the cytochromes c' are 3 orders of magnitude less reactive with flavodoxin semiquinone than are the c-type cytochromes. We interpret this result to be a consequence of the location of the exposed heme in cytochrome c' at the bottom of a deep groove in the surface of the protein, which is approximately 10-15 A deep and equally as wide. While free flavins are small enough to enter the groove, the flavin mononucleotide (FMN) prosthetic group of flavodoxin is apparently prevented by steric constraints from approaching the heme more closely than approximately 10 A without dynamic structural rearrangements. Most cytochromes c' are dimeric, but a few are monomeric. The three-dimensional structure of the Rhodospirillum molischianum cytochrome c' dimer suggests that the heme should be more exposed in the monomer than in the dimer, but no relationship is observed between intrinsic reactivity toward free flavin semiquinones and the aggregation state of the protein. Likewise, there is no evidence that the spin state or ligand field of the iron has any effect on intrinsic reactivity.(ABSTRACT TRUNCATED AT 250 WORDS)

Kinetics of reduction of high redox potential ferredoxins by the semiquinones of Clostridium pasteurianum flavodoxin and exogenous flavin mononucleotide. Electrostatic and redox potential effects.

We have measured the ionic strength dependence of the rate constants for the electron-transfer reactions of flavin mononucleotide (FMN) and flavodoxin semiquinones with 10 high redox potential ferredoxins (HiPIP's). The rate constants were extrapolated to infinite ionic strength by using a theoretical model of electrostatic interactions developed in our laboratory. In all cases, the sign of the electrostatic interaction was the same as the protein net charge, but the magnitudes were much smaller. The results are consistent with a model in which the electrical charges are approximately uniformly distributed over the HiPIP surface and in which there are both short- and long-range electrostatic interactions. An electrostatic field calculation for Chromatium vinosum HiPIP is consistent with this. The presumed site of electron transfer includes that region of the protein surface to which the iron-sulfur cluster is nearest and appears to be relatively hydrophobic. The principal short-range electrostatic interaction would involve the negative charge on the iron-sulfur cluster. For some net negatively charged proteins, this effect is magnified, and for net positively charged HiPIP's, it is counterbalanced. The rate constants extrapolated to infinite ionic strength can be correlated with redox potential differences between the reactants, as has previously been shown for cytochrome-flavin semiquinone reactions. Both electrostatic and redox potential effects are magnified for the flavodoxin semiquinone as compared to the FMN semiquinone-HiPIP reactions. This was also observed previously for the flavin semiquinone-cytochrome reactions.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

Resonance raman spectra of flavin semiquinones stabilized by N5 methylation

Resonance Raman spectra are reported for the semiquinone of N5-methyl derivatives of FMN (flavin mononucleotide) in H 2O and 2H 2O, 8-chloro FMN and FAD (flavin adenine dinucleotide) with 647.1 nm excitation, in the first π-π ∗ absorption band, using KI to quench fluorescence. The spectral pattern is similar to that of oxidized flavin, in its first absorption band, with appreciable shifts, up to approx. 50 cm −1, in corresponding frequencies. There are also significant shifts with respect to the previously reported resonance Raman spectrum of flavodoxin semiquinone, reflecting the substitution of CH 3 for H at N5. The N5-methyl FAD semiquinone spectrum is also reported for 514.5 nm excitation, in resonance with the second π-π ∗ transition. The intensity pattern is quite different, the spectrum being dominated by a band at 1611 cm −1, assigned to a mode localized primarily on the central pyrazine ring.

Transient kinetics of redox reactions of flavodoxin: effects of chemical modification of the flavin mononucleotide prosthetic group on the dynamics of intermediate complex formation and electron transfer.

The effects of structural modifications of the flavin mononucleotide (FMN) prosthetic group of Clostridium pasteurianum flavodoxin on the kinetics of electron transfer to the oxidized form (from 5-deazariboflavin semiquinone produced by laser flash photolysis) and from the semiquinone form (to horse heart cytochrome c by using stopped-flow spectrophotometry) have been investigated. The analogues used were 7,8-dichloro-FMN, 8-chloro-FMN, 7-chloro-FMN, and 5,6,7,8-tetrahydro-FMN. The ionic strength dependence of cytochrome c reduction was not affected by chlorine substitution, although the specific rate constants for complex formation and decay were appreciably smaller. On the other hand, all of the chlorine analogues had the same rate constant for deazariboflavin semiquinone oxidation. The rate constants for tetrahydro-FMN flavodoxin semiquinone reduction of cytochrome c were considerably smaller than those for the native protein. The implications of these results for the electron-transfer mechanism of flavodoxin are discussed.

Transient kinetics of electron transfer reactions of flavodoxin: ionic strength dependence of semiquinone oxidation by cytochrome c, ferricyanide, and ferric ethylenediaminetetraacetic acid and computer modeling of reaction complexes.

Electron transfer reactions between Clostridum pasteurianum flavodoxin semiquinone and various oxidants [horse heart cytochrome c, ferricyanide, and ferric ethylenediaminetetraacetic [horse heart cytochrome c, ferricyanide, and ferric ethylenediaminetetraacetic acid (EDTA)] have been studied as a function of ionic strength by using stopped-flow spectrophotometry. The cytochrome c reaction is complicated by the existence of two cytochrome species which react at different rates and whose relative concentrations are ionic strength dependent. Only the faster of these two reactions is considered here. At low ionic strength, complex formation between cytochrome c and flavodoxin is indicated by a leveling off of the pseudo-first-order rate constant at high cytochrome c concentration. This is not observed for either ferricyanide or ferric EDTA. For cytochrome c, the rate and association constants for complex formation were found to increase with decreasing ionic strength, consistent with negative charges on flavodoxin interacting with the positively charged cytochrome electron transfer site. Both ferricyanide and ferric EDTA are negatively charged oxidants, and the rate data respond to ionic strength changes as would be predicted for reactants of the same charge sign. These results demonstrate that electrostatic interactions involving negatively charged groups are important in orienting flavodoxin with respect to oxidants during electron transfer. We have also carried out computer modeling studies of putative complexes of flavodoxin with cytochrome c and ferricyanide, which relate their structural properties to both the observed kinetic behavior and some more general features of physiological electron transfer processes. The results of this study are consistent with the ionic strength behavior described above.