Flavodoxins

Medina and Cammack also performed three-pulse ESEEM on this species a prominent hfc thought to be due to N1 or N3 was detected. They were also able to observe small changes to the frequency-domain spectrum when substrate was bound to the enzyme. 

6 Flavodoxins. Flavodoxins are small, 36 kDa, flavoproteins containing one non-covalently bound FMN as cofactor. They are synthesized by cyanobacteria when the culture medium is iron deficient and replace ferredoxin in electron-transfer reactions including reduction of nitrogenase in Klebsiella and Azotobacter and electron transfer from photosynthetic membranes to fer-redoxin-NADP+ reductase. 

On binding to the apoflavodoxin the midpoint redox potentials of the FMN are drastically altered and the FMN semiquinone becomes much more stable, thus allowing flavodoxin to function as a one-electron transfer centre in which the flavin cycles between the semiquinone and fully reduced oxidation states. An interesting aspect of cofactor binding is that in most flavodoxins the isoalloxazine ring is sandwiched between two aromatic residues one is a highly conserved tyrosine (Y94) and the other usually a tryptophan (W57). 

Borruel and co-workers used EPR spectroscopy to monitor the expresion of holoflavodoxin from cyanobacterium Anabaena PCC 7119 in E. coli It could be shown by EPR, on whole cells frozen in 1 1 glycerol water mixtures, that complete synthesis of the holoprotein takes much longer than that of the apoprotein, and thus the optimal harvest time after induction, 10 hours in this case, could be determined. 

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Purified by paper chromatography using /crt-butanol-water, cutting out the main spot and eluting with water. Also purified by adsorption onto an apo-flavodoxin column, followed by elution and freeze drying Crystd from acidic aqueous soln. [Mayhew and Strating Eur J Biochem 59 539 1976.]... 

Figure 1 The basis of comparative protein structure modeling. Comparative modeling is possible because evolution resulted in families of proteins, such as the flavodoxin family, modeled here, which share both similar sequences and 3D structures. In this illustration, the 3D structure of the flavodoxin sequence from C. crispus (target) can be modeled using other structures in the same family (templates). The tree shows the sequence similarity (percent sequence identity) and structural similarity (the percentage of the atoms that superpose within 3.8 A of each other and the RMS difference between them) among the members of the family.

Figure 2.11 Beta sheets are usuaiiy represented simply by arrows in topology diagrams that show both the direction of each (3 strand and the way the strands are connected to each other along the polypeptide chain. Such topology diagrams are here compared with more elaborate schematic diagrams for different types of (3 sheets, (a) Four strands. Antiparallel (3 sheet in one domain of the enzyme aspartate transcarbamoylase. The structure of this enzyme has been determined to 2.8 A resolution in the laboratory of William Lipscomb, Harvard University, (b) Five strands. Parallel (3 sheet in the redox protein flavodoxin, the structure of which has been determined to 1.8 A resolution in the laboratory of Martha Ludwig, University of Michigan, (c) Eight strands. Antiparallel barrel in the electron carrier plastocyanln. This Is a closed barrel where the sheet is folded such that (3 strands 2 and 8 are adjacent. The structure has been determined to 1.6 A resolution in the laboratory of Hans Freeman in Sydney, Australia. (Adapted from J. Richardson.)...

The first structure, flavodoxin (Figure 4.14a), has one such position, between strands 1 and 3. The connection from strand 1 goes to the right and that from strand 3 to the left. In the schematic diagram in Figure 4.14a we can see that the corresponding a helices are on opposite sides of the p sheet. The loops from these two p strands, 1 and 3, to their respective a helices form the major part of the binding cleft for the coenzyme FMN (flavin mononucleotide). 

FIGURE 6.24 (a) The alpha helix consisting of residues 153-166 (red) in flavodoxin from Anahaena is a surface helix and is amphipathic. (b) The two helices (yellow and blue) in the interior of the citrate synthase dimer (residues 260-270 in each monomer) are mostly hydrophobic, (c) The exposed helix (residues 74-87—red) of calmodulin is entirely accessible to solvent and consists mainly of polar and charged residues. 

In globular protein structures, it is common for one face of an a-helix to be exposed to the water solvent, with the other face toward the hydrophobic interior of the protein. The outward face of such an amphiphilic helix consists mainly of polar and charged residues, whereas the inward face contains mostly nonpolar, hydrophobic residues. A good example of such a surface helix is that of residues 153 to 166 of flavodoxin from Anabaena (Figure 6.24). Note that the helical wheel presentation of this helix readily shows that one face contains four hydrophobic residues and that the other is almost entirely polar and charged. 

For flavodoxin in Figure 6.32, identify the right-handed crossovers and the left-handed cross-overs in the parallel /3-sheet. 

Pyruvate flavodoxin oxidoreductase Flavodoxin electron donor to nitrogenase... 

However, some data have been more difficult to incorporate into the mechanism shown in Figs. 8 and 9. As reported 21) in Section II,B the Fe protein can be reduced by two electrons to the [Fe4S4]° redox state. In this state the protein is apparently capable of passing two electrons to the MoFe protein during turnover, although it is not clear whether dissociation was required between electron transfers. More critically, it has been shown that the natural reductant flavodoxin hydroquinone 107) and the artificial reductant photoexcited eosin with NADH 108) are both capable of passing electrons to the complex between the oxidized Fe protein and the reduced MoFe protein, that is, with these reductants there appears to be no necessity for the complex to dissociate. Since complex dissociation is the rate-limiting step in the Lowe-Thorneley scheme, these observations could indicate a major flaw in the scheme. 

Some aspects of the Lowe-Thomeley mechanism for nitrogenase action, which has served us well over the past 15 years, are being called into question. In particular, the necessity for protein-protein dissociation after each electron transfer, the rate-determining step with dithionite as reductant, is being questioned when the natural electron donor flavodoxin or other artificial systems are used. Some aspects of the mechanism should be reinvestigated. 

Moreover, an electron transfer chain could be reconstituted in vitro that is able to oxidize aldehydes to carboxylic acids with concomitant reduction of protons and net production of dihydrogen (213, 243). The first enzyme in this chain is an aldehyde oxidoreductase (AOR), a homodimer (100 kDa) containing one Mo cofactor (MOD) and two [2Fe—2S] centers per subunit (199). The enzyme catalytic cycle can be regenerated by transferring electrons to flavodoxin, an FMN-con-taining protein of 16 kDa (and afterwards to a multiheme cytochrome and then to hydrogenase) ... 

This study of such of an electron transfer chain is most timely, since the 3D structures of all the components involved are known (and related components can easily be obtained by homology molecular modeling). Proposals of structural models for the complexes formed between D. gigas AOR and flavodoxin, based on the available X-ray... 

This review will not be concerned with functionally alternative structures and metabolites which appear in iron-limited growth. Thus Clostridium pasteurianum and other bacteria when grown in the presence of iron form ferredoxin grown at low iron the same organisms form flavodoxin, a flavoprotein [Knight, ., Jr., Hardy, R. W. J. Biol. Chem. 242, 1370 (1967) Mayhem, S. G., Massey, V. J. Biol. Chem. 244, 794 (1969)]. 

Mayhew, S.G. 1978. The redox potential of dithionite and SO,- from equilibrium reactions with flavodoxins, methyl viologen and hydrogen plus hydrogenase. European Journal of Biochemistry 85 535-547. 

Reduction of Fe-protein by electron carriers such as ferredoxin or flavodoxin or by inorganic dithionite ion. 

Researchers studying the stepwise kinetics of nitrogenase electron transfer using stopped-flow kinetic techniques have presented other scenarios. One hypothesis presents kinetic evidence that dissociation of Fe-protein from MoFe-protein is not necessary for re-reduction of Fe-protein by flavodoxins.13 These authors state that the possibility of ADP-ATP exchange while Fe-protein and MoFe-protein are complexed with each other cannot be excluded and that dissociation of the complex during catalysis may not be obligatory when flavodoxin is the Fe-protein reductant. This leads to the hypothesis that MgATP binds to the preformed Fe-protein/... 

The best characterized B 12-dependent methyltransferases is methionine synthase (Figure 15.11) from E. coli, which catalyses the transfer of a methyl group from methyltetrahydrofolate to homocysteine to form methionine and tetrahydrofolate. During the catalytic cycle, B12 cycles between CH3-Co(in) and Co(I). However, from time to time, Co(I) undergoes oxidative inactivation to Co(II), which requires reductive activation. During this process, the methyl donor is S-adenosylmethionine (AdoMet) and the electron donor is flavodoxin (Fid) in E. coli, or methionine synthase reductase (MSR) in humans. Methionine synthase... 

With incorporation of one atom of oxygen 1.19 Acting on reduced flavodoxin as donor... 

Fig. 21. An example of parallel /9 sheet, from flavodoxin (residues 82-86, 49-53, and 2-6). In the pattern characteristic of parallel j9 sheet, the hydrogen bonds are evenly spaced but slanted in alternate directions. Since both sides of the sheet are covered by other main chain (as is almost always true for parallel sheet), side groups pointing in both directions are predominantly hydrophobic except at the ends of the strands.

Fig. 23. Schematic drawing of the backbone of flavodoxin, a protein in which a parallel 0 sheet is the dominant structural feature. The sheet (represented by arrows) is shown from one edge, so that the characteristic twist can be seen clearly.

Fig. 25. A topological schematic diagram of the connectivity in the parallel /3 sheet of flavodoxin. Arrows represent the /3 strands thin-line connections lie below the plane of the sheet and fat connections above it. No attempt is made to indicate the length or conformation of the connecting chains (most of them are helical) or the twist of the fi sheet. The topology can also be specified by a sequential list of the connection types in this case, - lx,+ 2x,+ lx,+ lx.

Doubly wound variations Glyceraldehyde-phosphate dehydrogenase domain 1 Phosphorylase domain 1, central three layers Flavodoxin... 

The doubly wound structures were first recognized as a category by Rossmann in comparing flavodoxin with lactate dehydrogenase dl. As more and more protein structures were solved which fell into this... 

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