Stephen G. Mayhew

Separation of hydrogenase from intact cells of Desulfovibrio vulgaris Purification and properties

(Hildenborough [l-3] and Miyazaki [4,5] ). In most of the purification procedures that have been described the bacteria were broken mechanically, and in the case of the Miyazaki strain, a subsequent treatment with pancreatin or trypsin was found to be required to solubilize the enzyme [4,5]. The recoveries of activity after purification were low (0.4-2.8%), and purified preparations from a single strain differed in a number of properties, including molecular weight, subunit composition and iron content [2,3]. Part of the hydrogenase activity in a related organism,

2 Flavodoxins and Electron-Transferring Flavoproteins

Publisher Summary This chapter discusses two classes of flavoproteins that function solely to mediate electron transfer between the prosthetic groups of other proteins. According to the function the protein was termed electron-transferring flavoprotein. More recently, a different class of flavoprotein carriers has been isolated from microorganisms.These proteins have been called “flavodoxins” because of their functional interchangeability with the ferredoxins. The chapter describes the flavodoxins because they are the first flavoproteins for which three-dimensional structures have been determined. Organisms from which flavoproteins of the flavodoxin type have been isolated include several strictly anerobic bacteria, representatives from the obligately aerobic, facultatively anaerobic, and photosynthetic. Flavodoxins do not react directly with small molecules, such as the pyridine nucleotides, and their only known biochemical substrates are other redox proteins. The relative activities of flavodoxins and ferredoxins as electron carriers have been determined in both plant and microbial systems with results, which indicate that transfer rates vary somewhat according to the source of the carrier.

Cloning and Analysis of the Genes for a Novel Electron-transferring Flavoprotein from Megasphaera elsdenii EXPRESSION AND CHARACTERIZATION OF THE RECOMBINANT PROTEIN

The genes that encode the two different subunits of the novel electron-transferring flavoprotein (ETF) fromMegasphaera elsdenii were identified by screening a partial genomic DNA library with a probe that was generated by amplification of genomic sequences using the polymerase chain reaction. The cloned genes are arranged in tandem with the coding sequence for the β-subunit in the position 5′ to the α-subunit coding sequence. Amino acid sequence analysis of the two subunits revealed that there are two possible dinucleotide-binding sites on the α-subunit and one on the β-subunit. Comparison of M. elsdenii ETF amino acid sequence to other ETFs and ETF-like proteins indicates that while homology occurs with the mitochondrial ETF and bacterial ETFs, the greatest similarity is with the putative ETFs from clostridia and withfixAB gene products from nitrogen-fixing bacteria. The recombinant ETF was isolated from extracts of Escherichia coli. It is a heterodimer with subunits identical in size to the native protein. The isolated enzyme contains approximately 1 mol of FAD, but like the native protein it binds additional flavin to give a total of about 2 mol of FAD/dimer. It serves as an electron donor to butyryl-CoA dehydrogenase, and it also has NADH dehydrogenase activity.

Cloning, Overexpression, and Characterization of Peroxiredoxin and NADH Peroxiredoxin Reductase from Thermus aquaticus

The genes for peroxiredoxin (Prx) and NADH:peroxiredoxin oxidoreductase (PrxR) have been cloned from the thermophilic bacterium Thermus aquaticus. prxis located upstream from prxR, the two genes being separated by 13 bases. The amino acid sequences show that Prx is related to two-cysteine peroxiredoxins from a range of organisms and that PrxR resembles NADH-dependent flavoenzymes that catalyze the reduction of peroxiredoxins in mesophilic bacteria. The sequence of PrxR also resembles those of thioredoxin reductases (TrxR) from thermophiles but with an N-terminal extension of about 200 residues. PrxR has motifs for two redox-active disulfides, one in the FAD-binding site, as occurs in TrxR, and the other in the N-terminal extension. The molecular masses of the monomers of Prx and PrxR are 21.0 and 54.9 kDa, respectively; both enzymes exist as multimers. The recombinant flavoenzyme requires 3 mol equivalents of dithionite for full reduction, as is consistent with 1 FAD and 2 disulfides per monomer. PrxR and Prx together catalyze the anaerobic reduction of hydrogen peroxide. The activity of Prx is much less than has been observed with homologous proteins. Prx appears to be inactivated by cumene hydroperoxide. PrxR itself has low peroxidase activity.

Structure and mechanism of bacterial hydrogenase

Abstract Hydrogenases, a diverse group of Feue5f8S enzymes, catalyse the reversible oxidation of H 2 with a variety of electron carriers

A photo-CIDNP study of the active sites of Megasphaera elsdenii and Clostridium MP flavodoxins.

Megasphaera elsdenii and Clostridium MP flavodoxins have been investigated by photo‐CIDNP techniques. Using time‐resolved spectroscopy and external dyes carrying different charges it was possible to assign unambiguously the resonance lines in the NMR‐spectra to tyrosine, tryptophan and methionine residues in the two proteins. The results show that Trp‐91 in M.elsdenii and Trp‐90 in Cl.MP flavodoxin are strongly immobilized and placed directly above the benzene subnucleus of the prosthetic group. The data further indicate that the active sites of the two flavodoxins are extremely similar.

Backbone dynamics of oxidized and reduced D. vulgaris flavodoxin in solution

Recombinant Desulfovibrio vulgaris flavodoxin was produced inEscherichia coli. A complete backbone NMR assignment for the two-electronreduced protein revealed significant changes of chemical shift valuescompared to the oxidized protein, in particular for the flavinemononucleotide (FMN)-binding site. A comparison of homo- and heteronuclearNOESY spectra for the two redox states led to the assumption that reductionis not accompanied by significant changes of the global fold of the protein.The backbone dynamics of both the oxidized and reduced forms of D. vulgarisflavodoxin were investigated using two-dimensional15N-1H correlation NMR spectroscopy.T1, T2 and NOE data are obtained for 95%of the backbone amide groups in both redox states. These values wereanalysed in terms of the ’model-free‘ approach introduced by Lipari andSzabo [(1982) J. Am. Chem. Soc., 104, 4546-;4559, 4559-;4570]. Acomparison of the two redox states indicates that in the reduced speciessignificantly more flexibility occurs in the two loop regions enclosing FMN.Also, a higher amplitude of local motion could be found for the N(3)H groupof FMN bound to the reduced protein compared to the oxidized state.

Complex formation between ferredoxin and ferredoxin-NADP+ reductase from Anabaena PCC 7119: Cross-linking studies

Ferredoxin-NADP+ reductase and ferredoxin from the cyanobacterium Anabaena PCC 7119 have been covalently cross-linked by incubation with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide. The covalent adduct, which shows a molecular mass consistent with a 1:1 stoichiometry of the two proteins, maintains nearly 60% of the NADPH-cytochrome c reductase activity of the enzyme saturated with ferredoxin and this value is considerably higher than when equimolar amounts of both proteins are assayed. No ternary complexes with Anabaena flavodoxin or horse heart cytochrome c were formed, suggesting that the binding site on the enzyme is the same for ferredoxin and flavodoxin and that ferredoxin-NADP+ reductase and cytochrome c bind at a common site on ferredoxin. In the noncovalent complex, titrated at pH 7, the oxidation-reduction potential of ferredoxin becomes 15 mV more negative and that of ferredoxin-NADP+ reductase 27 mV more positive compared to the proteins alone. When covalently linked, the midpoint potential of the enzyme has a value similar to that in the noncovalent complex, while the ferredoxin potential is 20 mV more positive compared to ferredoxin alone. The changes in redox potentials have been used to estimate the dissociation constants for the interaction of the different redox forms of the proteins, based on the value of 1.21 microM calculated for the oxidized noncovalent complex.

Dual role of FMN in flavodoxin function: Electron transfer cofactor and modulation of the protein-protein interaction surface

Flavodoxin (Fld) replaces Ferredoxin (Fd) as electron carrier from Photosystem I (PSI) to Ferredoxin-NADP(+) reductase (FNR). A number of Anabaena Fld (AnFld) variants with replacements at the interaction surface with FNR and PSI indicated that neither polar nor hydrophobic residues resulted critical for the interactions, particularly with FNR. This suggests that the solvent exposed benzenoid surface of the Fld FMN cofactor might contribute to it. FMN has been replaced with analogues in which its 7- and/or 8-methyl groups have been replaced by chlorine and/or hydrogen. The oxidised Fld variants accept electrons from reduced FNR more efficiently than Fld, as expected from their less negative midpoint potential. However, processes with PSI (including reduction of Fld semiquinone by PSI, described here for the first time) are impeded at the steps that involve complex re-arrangement and electron transfer (ET). The groups introduced, particularly chlorine, have an electron withdrawal effect on the pyrazine and pyrimidine rings of FMN. These changes are reflected in the magnitude and orientation of the molecular dipole moment of the variants, both factors appearing critical for the re-arrangement of the finely tuned PSI:Fld complex. Processes with FNR are also slightly modulated. Despite the displacements observed, the negative end of the dipole moment points towards the surface that contains the FMN, still allowing formation of complexes competent for efficient ET. This agrees with several alternative binding modes in the FNR:Fld interaction. In conclusion, the FMN in Fld not only contributes to the redox process, but also to attain the competent interaction of Fld with FNR and PSI.

[31] Determination of FMN and FAD by fluorescence titration with apoflavodoxin

Publisher Summary This chapter discusses the determination of flavin mononucleotide (FMN) and flavin-adenine dinucleotide (FAD), by fluorescence titration, with apoflavodoxin. In assay method, the apoenzymes of flavodoxins isolated from certain anaerobic bacteria bind FMN rapidly and tightly to form complexes that are nonfluorescent, but they do not react with FAD or riboflavin. The FMN content of a test solution, therefore, can be determined from the end point of a fluorescence titration with a standard solution of apoflavodoxin. FAD is determined from the difference between the end point of this titration and that of a second titration, following the treatment of the test sample, with phosphodiesterase to hydrolyze FAD to FMN. Commercial preparations of FMN contain up to 32% flavin impurity that is not bound by apoflavodoxin from Megasphaera elsdenii . The FMN and FAD in mixtures of the two flavins can be accurately determined by the method discussed in the chapter when the total flavin concentration is l μM and the ratio FMN:FAD is between 9:l and 1:9. Similar results are obtained when the total flavin concentration is about 0.1 μM, but only when the FMN in the mixture is greater than about 20 nM.