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1. 1. The reactions of the oxidised and semiquinone forms of Peptostreptococcus elsdenii flavodoxin with sodium dithionite have been studied by stopped flow spectrophotometry. At low ionic strength where comproportionation of flavodoxin is slow compared with the overall rate of reduction, the reactions with an excess of dithionite follow first-order kinetics. Semiquinone is not detected during the reduction of oxidised flavodoxin, possibly because the semiquinone is reduced very much faster than the oxidised protein. 2. 2. The rates of reduction are proportional to the square root of the dithionite concentration. This observation suggests that the reducing species is not dithionite itself, but a dissociation product. It is proposed that the reducing species is SO2. The results support a mechanism in which the reduction of oxidised flavodoxin occurs by two successive one-electron transfers, and is limited by the rate of reduction to the semiquinone level. 3. 3. The reaction of oxidized flavodoxin and fully reduced flavodoxin to form semiquinone is very sensitive to ionic strength and the salt composition of the medium. Increasing ionic strength causes a marked increase in rate. The rate constant at zero ionic strength, calculated by extrapolation, was 100 M-1[middle dot]min-1 at 25 [deg]C.

The amino acid sequence of the flavodoxin has been determined except for a few residues. The protein contains 137 amino acid residues and the NH2- and COOH-terminal residues are methionine and alanine respectively. The protein contains two cysteine residues in positions 54 and 127. However, the cysteine residue in position 127 appears to be involved in binding FMN and therefore the active site is located near the COOH-terminal position of the protein. There are no aromatic amino acid residues in this region of the protein.

1. 1. NADH dehydrogenase (EC 1.6.99.3) has been purified from the strictly anaerobic rumen bacterium Peptostreptococcus elsdenii. The purified protein is specific for NADH as electron donor; it can use 2,6-dichlorophenolindophenol (DCIP), cytochrome c, K3FE(CN)6 or flavodoxin as electron acceptor. It has a molecular weight of about 63 000. The absorption spectrum shows maxima at 475, 375, 356, 318, and 273 nm. It has shoulders at 425, 460, and 510 nm. The protein is fluorescent with an emission maximum at 550 nm. 2. 2. The chromophore is released from the NADH dehydrogenase by either heat treatment or extraction with trichloroacetic acid. The free chromophore shows an increase of absorption in the visible spectrum and a shift of the maximum to 470 nm. The fluorescence emission maximum is shifted to 530 nm, but the intensity is unchanged. 3. 3. Hydrolysis of the free chromophore with phosphodiesterase causes a 12% increase in the visible absorption and a 2-fold increase in the fluorescence (exciting light at 480 nm). The chromatographic behavior is also affected by this treatment. 4. 4. The hydrolysed chromophore is bound by apoflavodoxin with 95% quenching of the fluorescence and an 18% decrease in the visible absorption. The complex of apoflavodoxin and the unknown chromophore is reduced by light irradiation in the presence of EDTA to an intermediate similar to flavodoxin semiquinone but with a distinctively different absorption spectrum. It is concluded that the chromophore in the NADH dehydrogenase is a flavin dinucleotide similar to FAD but modified in the isoalloxazine ring.

An anaerobic titration apparatus capable of titrations of 2.5 ml of solution with microliter quantities of titrant is described. Numerous samples can be titrated with the same stock solution and back titrations with different titrants are possible. No detectable O2 leaks (5 m[mu]moles of O2 or greater) were observed over a 6 day period. Biochemical applications of the apparatus are discussed.

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.

The reactions of excess apoflavodoxin from Desulfovibrio vulgaris, Anabaena variabilis and Azotobacter vinelandii with riboflavin 57prime;-phosphate (FMN), riboflavin 3′,5'′-bisphosphate and riboflavin are pseudo-first-order. The rates increase with decreasing pH in the range pH 5-8, and, in general, they increase with increasing ionic strength to approach a maximum at an ionic strength greater than 0.4 M. The rate of FMN binding in phosphate at high pH increases to a maximum at an ionic strength of about 0.1 M, and then decreases as the phosphate concentration is increased further. The dissociation constants for the complexes with FMN and riboflavin decrease with an increase of ionic strength. Inorganic phosphate stabilizes the complex with riboflavin. The effects of phosphate on riboflavin binding suggest that phosphate interacts with the apoprotein at the site normally occupied by the phosphate of FMN. Redox potentials determined for the oxidized/semiquinone and semiquinone/ hydroquinone couples of the riboflavin and FMN complexes were used with Kd values for the complexes with the oxidized flavins to calculate values for Kd for the semiquinone and hydroquinone complexes. The hydroquinone complexes are all less stable than the complexes with the two other redox forms of the flavin. Destabilization of the hydroquinone is less marked in the complexes with riboflavin, supporting a proposal that the terminal phosphate group of FMN plays a role in decreasing the stability of the hydroquinone complex and in decreasing the redox potential of the semiquinone/hydroquinone couple.

The reactions of excess apoflavodoxin from Desulfovibrio vulgaris, Anabaena variabilis and Azotobacter vinelandii with ribo- flavin 5«-phosphate (FMN), riboflavin 3«,5«-bisphosphate and riboflavin are pseudo-first-order. The rates increase with decreasing pH in the range pH 5-8, and, in general, they increase with increasing ionic strength to approach a maximum at an ionic strength greater than 0.4 M. The rate of FMN binding in phosphate at high pH increases to a maximum at an ionic strength of about 0.1 M, and then decreases as the phosphate concentration is increased further. The dissociation constants for the complexes withFMNand riboflavin decrease with an increase of ionic strength. Inorganic phosphate stabilizes the complex with riboflavin. The effects of phosphate on riboflavin binding suggest that phosphate interacts with the apoprotein at the site normally occupied by the phosphate of FMN. Redox potentials determined for the oxidized}semiquinone and semiquinone} hydroquinone couples of the riboflavin and FMN complexes were used with Kd values for the complexes with the oxidized flavins to calculate values for Kd for the semiquinone and hydroquinone complexes. The hydroquinone complexes are all less stable than the complexes with the two other redox forms of the flavin. Destabilization of the hydroquinone is less marked in the complexes with riboflavin, supporting a proposal that the terminal phosphate group of FMN plays a role in decreasing the stability of the hydroquinone complex and in decreasing the redox potential of the semiquinone}hydroquinone couple.

The oxidation-reduction potentials of ferredoxin-NADP' reductase and flavodoxin from the cyanobacterium Anuhaena PCC 7119 were determined by potentiometry. The potentials at pH 7 for the oxidized flavodoxin/ flavodoxin semiquinone couple (E2) and the flavodoxin seniiquinone/hydroquinone couple (El) were - 21 2 mV and -436 mV, respectively. El 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 pK, 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 (A&, = - 30 mV/pH). In the 1 : 1 electrostatic complex of the two proteins titrated at pH 7, E2 was shifted by + 8 mV and El 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 El 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 Anaharna vuriahilis was complexed with flavodoxin from Azotnbacter 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.

Flavodoxins are small flavoproteins which contain a single molecule of FMN as Prosthetic group.