Detection of a tryptophan radical as an intermediate species in the reaction of horseradish peroxidase mutant (Phe-221 --> Trp) and hydrogen peroxide

The crucial reaction intermediate in the reaction of peroxidase with hydrogen peroxide (H2O2), compound I, contains a porphyrin pi-cation radical in horseradish peroxidase (HRP), which catalyzes oxidation of small organic and inorganic compounds, whereas cytochrome c peroxidase (CcP) has a radical center on the tryptophan residue (Trp-191) and oxidizes the redox partner, cytochrome c. To investigate the roles of the amino acid residue near the heme active center in discriminating the function of the peroxidases in these two enzymes, we prepared a CcP-like HRP mutant, F221W (Phe-221 --> Trp). Although the rapid spectral scanning and stopped-flow experiments confirmed that the F221W mutant reacts with H2O2 to form the porphyrin pi-cation radical at the same rate as for the wild-type enzyme, the characteristic spectral features of the porphyrin pi-cation radical disappeared rapidly, and were converted to the compound II-type spectrum. The EPR spectrum of the resultant species produced by reduction of the porphyrin pi-cation radical, however, was quite different from that of compound II in HRP, showing typical signals from a Trp radical as found for CcP. The sequential radical formation from the porphyrin ring to the Trp residue implies that the proximal Trp is a key residue in the process of the radical transfer from the porphyrin ring, which differentiates the function of peroxidases.     

Interaction of NADPH-adrenodoxin reductase with NADP+ as studied by pulse radiolysis

The reduction of flavin in NADH--adrenodoxin reductase by the hydrated electron (eaq-) was investigated by pulse radiolysis. The eaq- reduced directly the flavin of the reductase to form a blue semiquinone of the enzyme. Subsequently, the semiquinone decayed by dismutation to form the oxidized and fully reduced forms of the enzyme with a second-order rate constant of 4.4 x 10(4) M-1 s-1. In the presence of equimolar NADP+, the decay of eaq- accompanied an absorption increase at 400 nm, the spectrum of which, formed transiently, is identical to that of NADP radical (NADP.). Subsequently, the transient species decayed concomitantly with the formation of the semiquinone. The rate constant in the formation of the semiquinone was independent of the concentration of the enzyme (6.1 x 10(4) s-1 at pH 7.5). From these results, it is concluded that eaq- reacts with NADP+ bound to the enzyme to form NADP. initially, and subsequently, an electron flows from the NADP. to the flavin by an intracomplex electron transfer. A similar result was obtained in the reaction of CO2- or N-methylnicotinamide radical with the NADP(+)-adrenodoxin reductase complex. These results suggest that the nicotinamide moiety of NADP+ bound to the enzyme is accessible to the solvent and masks the flavin completely.     

The soluble alpha-glycerophosphate oxidase from Enterococcus casseliflavus. Sequence homology with the membrane-associated dehydrogenase and kinetic analysis of the recombinant enzyme

The soluble flavoprotein alpha-glycerophosphate oxidase from Enterococcus casseliflavus catalyzes the oxidation of a "non-activated" secondary alcohol, in contrast to the flavin-dependent alpha-hydroxy- and alpha-amino acid oxidases. Surprisingly, the alpha-glycerophosphate oxidase sequence is 43% identical to that of the membrane-associated alpha-glycerophosphate dehydrogenase from Bacillus subtilis; only low levels of identity (17-22%) result from comparisons with other FAD-dependent oxidases. The recombinant alpha-glycerophosphate oxidase is fully active and stabilizes a flavin N(5)-sulfite adduct, but only small amounts of intermediate flavin semiquinone are observed during reductive titrations. Direct determination of the redox potential for the FAD/FADH2 couple yields a value of -118 mV; the protein environment raises the flavin potential by 100 mV in order to provide for a productive interaction with the reducing substrate. Steady-state kinetic analysis, using the enzyme-monitored turnover method, indicates that a ping-pong mechanism applies and also allows the determination of the corresponding kinetic constants. In addition, stopped-flow studies of the reductive half-reaction provide for the measurement of the dissociation constant for the enzyme. alpha-glycerophosphate complex and the rate constant for reduction of the enzyme flavin. These and other results demonstrate that this enzyme offers a very promising paradigm for examining the protein determinants for flavin reactivity and mechanism in the energy-yielding metabolism of alpha-glycerophosphate.     

Catalytic properties of adenylylsulfate reductase from Desulfovibrio vulgaris Miyazaki

Adenylylsulfate reductase (EC 1.8.99.2) isolated from Desulfovibrio vulgaris Miyazaki catalyzes electron transfer from dihydroflavin coenzyme (FADH2, FMNH2, or dihydroriboflavin) to adenylyl sulfate (APS), and catalyzes flavin-mediated oxidation of ferrocytochrome c3 with APS. The reaction with FAD as an electron mediator was markedly stimulated in the presence of menadione. Km of the enzyme was about 0.015 mM for riboflavin and FAD in the presence of menadione. Free flavin coenzyme was found to be the normal cellular constituent. These observations suggested that free flavin coenzyme may be a physiological electron carrier for APS reductase, and the enzyme may be called AMP, sulfite:flavin oxidoreductase. Km (APS) of this enzyme is lower than 1 microM. The enzyme is not inhibited by ATP and GTP, but was inhibited by AMP and sulfite. Its extremely low Km (APS) enables this enzyme to reduce any traces of cytosolic APS which is present only at micromolar concentration, and inhibition by sulfite makes this organism utilize an energetically favorable electron acceptor, sulfite, preferentially over APS which is produced from sulfate at the cost of ATP.     

Interaction of pyridoxal 5'-phosphate with tryptophan-139 at the subunit interface of dimeric D-amino acid transaminase

The crystal structure of dimeric bacterial D-amino acid transaminase shows that the indole rings of the two Trp-139 side chains face each other in the subunit interface about 10 angstroms from the coenzyme, pyridoxal 5'-phosphate. To determine whether it has a role in the catalytic efficiency of the enzyme or interacts with the coenzyme, Trp-139 has been substituted by several different types of amino acids, and the properties of these recombinant mutant enzymes have been compared to the wild-type enzyme. In the native wild-type holoenzyme, the fluorescence of one of the three Trp residues per monomer is almost completely quenched, probably due to its interaction with PLP since in the native wild-type apoenzyme devoid of PLP, tryptophan fluorescence is not quenched. Upon reconstitution of this apoenzyme with PLP, the tryptophan fluorescence is quenched to about the same extent as it is in the native wild-type enzyme. The site of fluorescence quenching is Trp-139 since the W139F mutant in which Trp-139 is replaced by Phe has about the same amount of fluorescence as the wild-type enzyme. The circular dichroism spectra of the holo and the apo forms of both the wild-type and the W139F enzymes in the far-ultraviolet show about the same degree of ellipticity, consistent with the absence of extensive global changes in protein structure. Furthermore, comparison of the circular dichroism spectrum of the W139F enzyme at 280 nm with the corresponding spectral region of the wild-type enzyme suggests a restricted microenvironment for Trp-139 in the latter enzyme. The functional importance of Trp-139 is also demonstrated by the finding that its replacement by Phe, His, Pro, or Ala gives mutant enzymes that are optimally active at temperatures below that of the wild-type enzyme and undergo the E-PLP --> E-PMP transition as a function of D-Ala concentration with reduced efficiency. The results suggest that a fully functional dimeric interface with the two juxtaposed indole rings of Trp-139 is important for optimal catalytic function and maximum thermostability of the enzyme and, furthermore, that there might be energy transfer between Trp-139 and coenzyme PLP.     

The rate of CD4 decline as a determinant of progression to AIDS independent of the most recent CD4 count. The Italian Seroconversion Study

Horse liver alcohol dehydrogenase contains two tryptophan residues per subunit, Trp-15 on the surface of the catalytic domain and Trp-314 buried in the interface between the subunits of the dimer. We studied the contributions of the tryptophans to fluorescence and catalytic dynamics by substituting Trp-314 with a leucine residue and making two compensatory mutations that were required to obtain a stable protein, leading to the triple mutant M303F-L308I-W314L enzyme. The substitutions increased by two- to sixfold the turnover numbers for ethanol oxidation, acetaldehyde reduction, and the dissociation constants of the coenzymes. The rate of the exponential burst phase for the transient oxidation of ethanol increased slightly, but the rate of dissociation of the enzyme-NADH complex still limited turnover of ethanol, as for wild-type enzyme. The three substitutions at the dimer interface apparently activate the enzyme by allowing more rapid conformational changes that accompany coenzyme binding, probably due to movement of the loop containing residues 293 to 298. The emission spectrum of M303F-L308I-W314L enzyme, which contains Trp-15, was redshifted compared to wild-type enzyme. Time-resolved fluorescence measurements with the triple mutant show that the decay of Trp-15 is dominated by a approximately 7-ns component. In the mutant enzyme with Trp-15 substituted with phenylalanine, the decay of Trp-314 is dominated by a approximately 4-ns component. Solute quenching data for wild-type enzyme and the mutants show that only Trp-15 is exposed to iodide and acrylamide, whereas Trp-314 is inaccessible. The luminescence properties of the tryptophan residues in the mutated enzymes are consistent with conclusions from studies of the wild-type enzyme [M. R. Eftink, 1992, Adv. Biophys. Chem. 2, 81-114].     

Myoglobin-induced oxidative damage: evidence for radical transfer from oxidized myoglobin to other proteins and antioxidants

Reaction of equine Fe(III) myoglobin with H2O2 gives rise to an Fe(IV)-oxo species at the heme center and protein (globin)-derived radicals. Studies have shown that there are two (or more) sites for the protein-derived radical: at tyrosine (Tyr-103) or tryptophan (Trp-14). The latter radical reacts rapidly with oxygen to give a Trp-derived peroxyl radical. The formation of both the tyrosine phenoxyl radical and the tryptophan-derived peroxyl species have been confirmed in the present study; the latter appears to be the major initial radical, with the phenoxyl radical appearing at longer reaction times, possibly via secondary reactions. We have investigated, by EPR spectroscopy, the reactivity of the Trp-14 peroxyl radical with amino acids, peptides, proteins, and antioxidants, with the aim of determining whether this species can damage other targets, i.e., whether intermolecular protein-to-protein radical transfer and hence chain-oxidation occurs, and the factors that control these reactions. Three amino acids show significant reactivity: Tyr, Trp, and Cys, with Cys the least efficient. Evidence has also been obtained for (inefficient) hydrogen abstraction at peptide alpha-carbon sites; this may result in backbone cleavage in the presence of oxygen. The myoglobin Trp-14 peroxyl radical has been shown to react rapidly with a wide range of proteins to give long-lived secondary radicals on the target protein. These reactions appear to mainly involve Tyr residues on the target protein, although evidence for reaction at Trp has also been obtained. Antioxidants (GSH, ascorbate, Trolox C, vitamin E, and urate) react with the myoglobin-derived peroxyl radical; in some cases antioxidant-derived radicals are detected. These reactions are only efficient at high antioxidant concentrations, suggesting that protein-to-protein damage transfer and protein chain-oxidation may occur readily in biological systems.     

On the domain structure of cytochrome P450 102 (BM-3): isolation and properties of a 45-kDa FAD/NADP domain

Cytochrome P450 102 is a catalytically self-sufficient monooxygenase isolated from barbiturate-induced Bacillus megaterium. The enzyme contains FAD, FMN, and heme in a single polypeptide chain of 1048 residues, and each of the cofactors is believed to be located in a separate domain. In the present study we have used exhaustive endogenous proteolysis to produce a 45 kDa fragment of the cytochrome. This fragment bound the 2',5'-adenosine diphosphate moiety of NADP(H) strongly, with approximately the same dissociation constant as in the native enzyme, and contained only FAD (0.93 equivalents per polypeptide, epsilon 453nm = 11,200 M-1cm-1). Reduction of the flavin by sodium dithionite proceeded quite slowly to yield FADH2, but no stable semiquinone species was produced upon air re-oxidation. In contrast, NADPH rapidly reduced this FAD/NADP(H) domain aerobically to produce the FADH. semiquinone radical. At a 75:1 molar ratio of the FAD/NADP(H) domain to the P450 102 heme domain, no laurate hydroxylase activity was observed. Gas-phase sequence analysis showed the presence of two major sequences beginning at Phe646 (403 residues, MW 45,033) and Asp652 (397 residues). These data are in agreement with the crystal structures of related enzymes and closely define the boundary of the FAD/NADP+ domain in P450 102.     

Quantitation of hepatitis G virus RNA in allogeneic bone marrow transplant recipients. Stem Cell Transplantation Team

In thermolysin, tryptophan 115 seems to be at the S2 subsite. Trp-115 was replaced with tyrosine, phenylalanine, leucine, and valine during site-directed mutagenesis in order to evaluate the role of Trp-115 in the proteolytic activity of thermolysin. The mutant enzymes with Tyr-115 or Phe-115 had as much proteolytic activity as the wild-type enzyme, but the other two mutant enzymes had no activity. We found earlier that the substitution of Trp-115 with alanine, glutamic acid, lysine, and glutamine causes the enzyme to lose all activity, so an aromatic amino acid at position 115 seems to be essential for thermolysin.     

Intramolecular electron transfer in yeast flavocytochrome b2 upon one-electron photooxidation of the fully reduced enzyme: evidence for redox state control of heme-flavin communication

Flavocytochrome b2, which has been fully reduced using L-lactate, can be rapidly oxidized by 1 equiv using the laser-generated triplet state of 5-deazariboflavin. Parallel photoinduced oxidation occurs at the reduced heme and at the fully reduced FMN (FMNH2) prosthetic groups of different enzyme monomers, producing the anion semiquinone of FMN and a ferric heme. Following the initial oxidation reaction, rapid intramolecular reduction of the ferric heme occurs with concomitant oxidation of FMNH2, generating the neutral FMN semiquinone. The observed rate constant for this intramolecular electron transfer is 2200 s-1, which is 1 order of magnitude larger than the turnover number under these conditions. A slower reduction of the heme prosthetic group also occurs with an observed rate constant of approximately 10 s-1, perhaps due to intersubunit electron transfer from reduced FMN to heme. The rapid intramolecular electron transfer between the FMNH2 and ferric heme is eliminated upon addition of excess pyruvate (Ki = 3.8 mM). This latter result indicates that pyruvate inhibition of catalytic turnover apparently can occur at the FMNH2-->heme electron transfer step. These results markedly differ from those previously obtained (Walker, M. C., & Tollin, G. (1991) Biochemistry 30, 5546-5555) and confirmed here for electron transfer within the one-electron reduced enzyme and for the effect of pyruvate binding, suggesting that intramolecular communication between the heme and flavin prosthetic groups can be controlled by the redox state of the enzyme and by ligand binding to the active site.     

Design of a ruthenium-cytochrome c derivative to measure electron transfer to the radical cation and oxyferryl heme in cytochrome c peroxidase

A new ruthenium-labeled cytochrome c derivative was designed to measure the actual rate of electron transfer to the Trp-191 radical cation and the oxyferryl heme in cytochrome c peroxidase compound I {CMPI(FeIV = O,R.+)}. The H39C,C102T variant of yeast iso-1-cytochrome c was labeled at the single cysteine residue with a tris (bipyridyl)ruthenium(II) reagent to form Ru-39-Cc. This derivative has the same reactivity with CMPI as native yCc measured by stopped-flow spectroscopy, indicating that the ruthenium group does not interfere with the interaction between the two proteins. Laser excitation of the 1:1 Ru-39-Cc-CMPI complex in low ionic strength buffer (2 mM phosphate, pH 7) resulted in electron transfer from RuII* to heme c FeIII with a rate constant of 5 x 10(5) s-1, followed by electron transfer from heme c Fe II to the Trp-191 indolyl radical cation in CMPI(FeIV = O,R*+) with a rate constant of k(eta) = 2 x 10(6) s-1. A subsequent laser flash led to electron transfer from heme c to the oxyferryl heme in CMPII-(FeIV = O,R) with a rate constant of k(etb) = 5000 s-1. The location of the binding domain was determined using a series of surface charge mutants of CcP. The mutations D34N, E290N, and A193F each decreased the values of k(eta) and k(etb) by 2-4-fold, consistent with the use of the binding domain identified in the crystal structure of the yCc-CcP complex for reduction of both redox centers [Pelletier, H., & Kraut, J. (1992) Science 258, 1748-1755]. A mechanism is proposed for reduction of the oxyferryl heme in which internal electron transfer in CMPII(FeIV = O,R) leads to the regeneration of the radical cation in CMPII-(FeIII,R*+), which is then reduced by yCcII. Thus, both steps in the complete reduction of CMPI involve electron transfer from yCcII to the Trp-191 radical cation using the same binding site and pathway. Comparison of the rate constant k(eta) with theoretical predictions indicate that the electron transfer pathway identified in the crystalline yCc-CcP complex is very efficient. Stopped-flow studies indicate that native yCcII initially reduces the Trp-191 radical cation in CMPI with a second-order rate constant ka, which increases from 1.8 x 10(8) M-1 s-1 at 310 mM ionic strength to > 3 x 10(9) M-1 s-1 at ionic strengths below 100 mM. A second molecule of yCcII then reduces the oxyferryl heme in CMPII with a second-order rate constant kb which increases from 2.7 x 10(7) M-1 s-1 at 310 mM ionic strength to 2.5 x 10(8) M-1 s-1 at 160 mM ionic strength. As the ionic strength is decreased below 100 mM the rate constant for reduction of the oxyferryl heme becomes progressively slower as the reaction is limited by release of the product yCcIII from the yCcIII-CMPII complex. Both ruthenium photoreduction studies and stopped-flow studies demonstrate that the Trp-191 radical cation is the initial site of reduction in CMPI under all conditions of ionic strength.     

Purification, characterization, and mechanism of a flavin mononucleotide-dependent 2-nitropropane dioxygenase from Neurospora crassa

A nitroalkane-oxidizing enzyme was purified to homogeneity from Neurospora crassa. The enzyme is composed of two subunits; the molecular weight of each subunit is approximately 40,000. The enzyme catalyzes the oxidation of nitroalkanes to produce the corresponding carbonyl compounds. It acts on 2-nitropropane better than on nitroethane and 1-nitropropane, and anionic forms of nitroalkanes are much better substrates than are neutral forms. The enzyme does not act on aromatic compounds. When the enzyme reaction was conducted in an 18O2 atmosphere with the anionic form of 2-nitropropane as the substrate, acetone (with a molecular mass of 60 Da) was produced. This indicates that the oxygen atom of acetone was derived from molecular oxygen, not from water; hence, the enzyme is an oxygenase. The reaction stoichiometry was 2CH3CH(NO2)CH3 + O2-->2CH3COCH3 + 2HNO2, which is identical to that of the reaction of 2-nitropropane dioxygenase from Hansenula mrakii. The reaction of the Neurospora enzyme was inhibited by superoxide anion scavengers in the same manner as that of the Hansenula enzyme. Both of these enzymes are flavoenzymes; however, the Neurospora enzyme contains flavin mononucleotide as a prosthetic group, whereas the Hansenula enzyme contains flavin adenine dinucleotide.     

Flavin mononucleotide-binding domain of the flavoprotein component of the sulfite reductase from Escherichia coli

The flavoprotein component (SiR-FP) of the sulfite reductase from Escherichia coli is an octamer containing one FAD and one FMN as cofactors per polypeptide chain. We have constructed an expression vector containing the DNA fragment encoding for the FMN-binding domain of SiR-FP. The overexpressed protein (SiR-FP23) was purified as a partially flavin-depleted polymer. It could incorporate FMN exclusively upon flavin reconstitution to reach a maximum flavin content of 1.2 per polypeptide chain. Moreover, the protein could stabilize a neutral air-stable semiquinone radical over a wide range of pHs. During photoreduction, the flavin radical accumulated first, followed by the fully reduced state. The redox potentials, determined at room temperature [E'1 (FMNH./FMN) = -130 +/- 10 mV and E'2 (FMNH2/FMNH.) = -335 +/- 10 mV], were very close to those previously reported for Salmonella typhimurium SiR-FP [Ostrowski, J., Barber, M. J., Rueger, D. C., Miller, B. E., Siegel, L. M., & Kredich, N. M. (1989) J. Biol. Chem. 264, 15796-15808]. Both the radical and fully reduced forms of SiR-FP23 were able to transfer their electrons to cytochrome c quantitatively. Altogether, the results presented herein demonstrate that the N-terminal end of E. coli SiR-FP forms the FMN-binding domain. It folds independently, thus retaining the chemical properties of the bound FMN, and provides a good model of the FAD-depleted form of native SiR-FP. Moreover, the FMN prosthetic group in SiR-FP23 and native SiR-FP is compared to that of cytochrome P450 reductase and bacterial cytochrome P450, which also contain one FAD and one FMN per polypeptide chain.     

Oxidation of phenolic compounds by lactoperoxidase. Evidence for the presence of a low-potential compound II during catalytic turnover

The lactoperoxidase (LPO)-catalyzed oxidation of p-phenols by hydrogen peroxide has been studied. The behavior of the enzyme differs from that of other peroxidases in this reaction. In particular LPO shows several catalytic intermediates during the catalytic cycle because of its capability to delocalize an oxidizing equivalent on a protein amino acid residue. In the phenol oxidation the enzyme Compound I species, containing an iron-oxo and a protein radical, uses the iron-oxo group at acidic pH and the protein radical in neutral or basic medium. Kinetic and spectroscopic studies indicate that the ionization state of an amino acid residue with pKa 5.8 +/- 0.2, probably the distal histidine, controls the enzyme intermediate forms at different pH. LPO undergoes inactivation during the oxidation of phenols. The inactivation is reversible and depends on the easy formation of Compound III even at low oxidant concentration. The inactivation is due to the substrate redox potential since the best substrate is that with lowest redox potential, while the worst substrate has the highest potential. This strongly indicates that Compound II, formed during catalytic turnover, has a low redox potential, making easier its oxidation by hydrogen peroxide to Compound III. The dependence of LPO activity on the phenols redox potential suggests that the protein radical where an oxidizing equivalent can be localized is a tyrosyl residue.     

Functional analysis by site-directed mutagenesis of individual amino acid residues in the flavin domain of Neurospora crassa nitrate reductase

Nitrate reductase of Neurospora crassa is a complex multi-redox protein composed of two identical subunits, each of which contains three distinct domains, an amino-terminal domain that contains a molybdopterin cofactor, a central heme-containing domain, and a carboxy-terminal domain which binds a flavin and a pyridine nucleotide cofactor. The flavin domain of nitrate reductase appears to have structural and functional similarity to ferredoxin NADPH reductase (FNR). Using the crystal structure of FNR and amino acid identities in numerous nitrate reductases as guides, site-directed mutagenesis was used to replace specific amino acids suspected to be involved in the binding of the flavin or pyridine nucleotide cofactors and thus important for the catalytic function of the flavin domain. Each mutant flavin domain protein was expressed in Escherichia coli and analyzed for NADPH: ferricyanide reductase activity. The effect of each amino acid substitution upon the activity of the complete nitrate reductase reaction was also examined by transforming each manipulated gene into a nit-3- null mutant of N. crassa. Our results identify amino acid residues which are critical for function of the flavin domain of nitrate reductase and appear to be important for the binding of the flavin or the pyridine nucleotide cofactors.     

Electrochemical and spectroscopic properties of the iron-sulfur flavoprotein from Methanosarcina thermophila

An iron-sulfur flavoprotein (Isf) from the methanoarchaeaon Methanosarcina thermophila, which participates in electron transfer reactions required for the fermentation of acetate to methane, was characterized by electrochemistry and EPR and M?ssbauer spectroscopy. The midpoint potential (Em) of the FMN/FMNH2 couple was -0.277 V. No flavin semiquinone was observed during potentiometric titrations; however, low amounts of the radical were observed when Isf was quickly frozen after reaction with CO and the CO dehydrogenase/acetyl-CoA synthase complex from M. thermophila. Isf contained a [4Fe-4S]2+/1+ cluster with g values of 2.06 and 1.93 and an unusual split signal with g values at 1.86 and 1.82. The unusual morphology was attributed to microheterogeneity among Isf molecules. The Em value for the 2+/1+ redox couple of the cluster was -0.394 V. Extracts from H2-CO2-grown Methanobacterium thermoautotrophicum cells catalyzed either the H2- or CO-dependent reduction of M. thermophila Isf. In addition, Isf homologs were found in the genomic sequences of the CO2-reducing methanoarchaea M. thermoautotrophicum and Methanococcus jannaschii. These results support a general role for Isf in electron transfer reactions of both acetate-fermenting and CO2-reducing methanoarchaea. It is suggested that Isf functions to couple electron transfer from ferredoxin to membrane-bound electron carriers, such as methanophenazine and/or b-type cytochromes.     

Energy transfer (deazaflavin-->FADH2) and electron transfer (FADH2-->T <> T) kinetics in Anacystis nidulans photolyase

DNA photolyases catalyze the photocycloreversion of cyclobutane pyrimidine dimers. The enzyme from the cyanobacterium Anacystis nidulans contains two chromophores, 1,5-dihydroflavin adenine dinucleotide (FADH2) and 7,8-didemethyl-8-hydroxy-5-deazariboflavin (8-HDF). The photophysical/photochemical reactions leading to DNA repair were investigated by using time-resolved and steady-state fluorescence spectroscopy. It was found that the excited singlet state of 8-HDF transfers energy to FADH2 at a rate of 1.9 x 10(10) s-1 and a quantum yield of 0.98. Using the Forster equation for long-range energy transfer and assuming random orientations of the donor and acceptor the interchromophore distance was calculated to be 15 A. The excited singlet FADH2 which forms either by energy transfer from 8-HDF or by direct absorption of a photon has a lifetime of 1.8 ns in the absence of substrate and 0.14 ns in the presence of the photodimer indicating electron transfer from the FADH2 excited singlet state to the dimer at a rate of 6.5 x 10(9) s-1 and quantum efficiency of 92%.     

Reinterpretation of luminiscence properties of neurotoxins from the venom of Middle-Asian corba Naja oxiana eichw

A new interpretation of previous work (Bukolova-Orlova, T. G., Burstein, E.A. and Yukelson, L. Ya (1974) Biochim. Biophys. Acta 342, 272-280) and some new data on the luminescence of neurotoxins I and II from Naja oxiana venom is given, based on the newer data on their complete amino acid sequences. Very effective excitation energy exchange exists between Trp-27 and Trp-33 in neurotoxin I and between Trp-27 and Trp-28 in neurotoxin II, Which results in the tryptophanyl fluorescence spectra of each of the proteins seeming to be monocomponent ones. The lowered fluorescence quantium yield value, the shortened phosphorescence lifetime (80% of the emission has tau p less than 0.5 s, 20% has tau p = 4.8 s, comparing with usual tau p = 5.5-5.9 s) and decreased phosphorescence to fluorescence ratio (0.042, as compared to the usual 0.4-0.7) for neurotoxin I suggest that the indole chromophore of Trp-27 and/or Trp-33 are in contact with heavy sulfur atoms of disulfide, most probably of Cys(28)-Cys(32). Tryptophanyls in neurotoxin II are exposed to the solvent, however their accessibility in relation to that of the free tryptophan to the negatively charged quencher I- (0.455) is much higher than that for the positively charged Cs+ (0.08), which is probably due to the proximity of cationic Lys-25, Lys-26 and His-31. The difference of accessibility to the negative and positive quenchers is even more pronounced in the case of the neurotoxin I (1.04 and 0 +/- 0.02, respectively), though in its chromophore vicinity along the primary structure there is only one cationic group, Lys-25. This fact together with the analysis of the amino acid sequence, suggest that the space folding of this polypeptide results in the close proximity of Trp-27 and/or Trp-33 with the C-terminal peptide segment 67-73, which contains four cationic groups (His-67, Lys-69, Lys-71 and Arg-72).     

Design, synthesis, and characterization of a photoactivatable flavocytochrome molecular maquette

We report the construction of a synthetic flavo-heme protein that incorporates two major physiological activities of flavoproteins: light activation of flavin analogous to DNA photolyase and rapid intramolecular electron transfer between the flavin and heme cofactors as in several oxidoreductases. The functional tetra-alpha-helix protein comprises two 62-aa helix-loop-helix subunits. Each subunit contains a single cysteine to which flavin (7-acetyl-10-methylisoalloxazine) is covalently attached and two histidines appropriately positioned for bis-his coordination of heme cofactors. Both flavins and hemes are situated within the hydrophobic core of the protein. Intramolecular electron transfer from flavosemiquinone generated by photoreduction from a sacrificial electron donor in solution was examined between protoporphyrin IX and 1-methyl-2-oxomesoheme XIII. Laser pulse-activated electron transfer from flavin to meso heme occurs on a 100-ns time scale, with a favorable free energy of approximately -100 meV. Electron transfer from flavin to the lower potential protoporphyrin IX, with an unfavorable free energy, can be induced after a lag phase under continuous light illumination. Thus, the supporting peptide matrix provides an excellent framework for the positioning of closely juxtaposed redox groups capable of facilitating intramolecular electron transfer and begins to clarify in a simplified and malleable system the natural engineering of flavoproteins.     

Review of Rett syndrome

A FAD and [4Fe-4S]cluster-containing enzyme from Clostridium aminobutyricum catalyses the reversible dehydration of 4-hydroxybutyryl-CoA to crotonyl-CoA which involves the cleavage of an unactivated C-H bond at the beta-carbon. Transient oxidation of the substrate to an enoxy radical by FAD might facilitate the removal of this beta-proton, whereas no function could be attributed to the [4Fe-4S]cluster. In this paper the organic radical, which is formed by partial reduction of the enzyme with dithionite, was characterised as the neutral flavin semiquinone by EPR spectroscopy in H2O and D2O. The rapid electron-spin relaxation of the flavin semiquinone suggested a magnetic interaction with the [4Fe-4S]cluster. In order to obtain highly resolved information about nuclear spins in the vicinity of this paramagnetic centre, ENDOR spectroscopy was applied. The spectra were compared with those of the neutral semiquinone radicals of ferredoxin-NADP reductase and flavodoxin as well as with that of the anionic semiquinone radical of cholesterol oxidase. All ENDOR spectra showed strong couplings to the 8-methyl protons and to H-6 of the flavin. On addition of the substrates to the corresponding enzymes, the electron density changed significantly only at the 8-position. It decreased in the case of cholesterol oxidase and ferredoxin-NADP reductase, whereas an increase was observed with 4-hydroxybutyryl-CoA dehydratase. The results indicate an interaction of 4-hydroxybutyryl-CoA with the flavin as required by the proposed mechanism. Furthermore, the shift of electron density towards the benzoid ring of FAD in the dehydratase might be due to the location of the [4Fe-4S]cluster next to the 8-position as known from structurally characterised iron-sulfur flavoproteins.