Base pairing of adenine and uracil derivatives in the presence of water.

The association of chloroform-soluble derivatives of uracil and adenine has been examined in chloroform solution in the presence of dissolved water. Analysis by infrared spectroscopy shows that complex formation still occurs in these conditions, and that the extent of association is substantially unchanged by the presence of water. Evidence is presented for the coexistence of two kinds of base pair (involving, respectively, the C₂ and C₄ carbonyl groups of the pyrimidine) in the solutions, and for some displacement in their relative balance by the added water. The binding of water to the C₂ and C₄ carbonyl groups can be separately observed in both the free uracil derivative and its 1:1 complex with 9-ethyladenine. Little or no competition has been found to occur between the formation of base pairs and binding of water to the bases, as judged by measurements of water solubility in chloroform solutions of the bases individually and in 1:1 mixtures. The evidence suggests that this phenomenon can be largely explained by the formation of double hydrogen bonds by the uracil carbonyl groups. Taken together with recent published observations, the results indicate that hydrogen bonding may make a much greater energetic contribution to conformational stability of biopolymers in aqueous solution that has been supposed.     

Thermodynamics of the binding of flavin adenine dinucleotide to D-amino acid oxidase.

The enthalpy of binding, deltaHb, of flavin adenine dinucleotide to the apoenzyme of D-amino acid oxidase was determined by flow calorimetry at pH 8.5 to be +3.8, -4.1 and -11.0 kcal mol-1 at 10 degrees, 25 degrees and 38 degrees, respectively. These values correspond to a heat capacity change, deltaCp, of -530 cal K-1 mol-1. From the binding constant reported by Dixon and Kleppe (1965a) and the above enthalpies, the standard free energy and standard entropy of binding are evaluated. These thermodynamic data are interpreted in terms of hydrophobic and vibrational contributions (Sturtevant, 1977). The product of the assay reaction (Fonda and Anderson, 1967), benzoylformic acid, is a non-competitive inhibitor of the enzyme with a value for KI of 1.4 X 10(-4)M at 25 degrees.     

Characterisation of the flavin adenine dinucleotide binding region of Myxococcus xanthus protoporphyrinogen oxidase

Protoporphyrinogen oxidase (PPOX), the penultimate enzyme in the haem biosynthetic pathway catalysers the six electron oxidation of protoporphyrinogen-IX to protoporphyrin-IX, in the presence of flavin adenine dinucleotide (FAD) and oxygen. In humans, partial defects in PPOX result in variegate porphyria. In this study, the FAD binding region in Myxococcus xanthus PPOX was analysed by engineering and characterising a selection of mutant proteins. Amino acid residues which interact with FAD via their side chains were selected for study. Mutants were characterised and compared with wild type protein. Characterisation included FAD quantitation, analysis of FAD spectra and kinetic assay. Results revealed that Serine 20 mutants could still bind FAD, but polarity in this position is favourable, yet not essential for the integrity of FAD binding. Study of Glutamate 39 mutants suggest that a negative charge at position 39 is clearly favoured for interaction with the ribose ring of FAD, as all non-conservative replacements could not bind sufficient FAD. Asparagine 441 appears not to be directly involved in FAD binding but rather in stabilizing the FAD, and polarity in this position appears important. Tryptophan 408 may play a role in orientating or stabilizing the bound substrate during catalysis, and a non-polar (or slightly polar) residue is favoured at this position; however, aromaticity in this position appears not to be critical. Overall this study sheds further light on how M. xanthus PPOX interacts with FAD.     

Partially folded structure of flavin adenine dinucleotide-depleted ferredoxin-NADP+ reductase with residual NADP+ binding domain

Maize ferredoxin-NADP(+) reductase (FNR) consists of flavin adenine dinucleotide (FAD) and NADP(+) binding domains with a FAD molecule bound noncovalently in the cleft between these domains. The structural changes of FNR induced by dissociation of FAD have been characterized by a combination of optical and biochemical methods. The CD spectrum of the FAD-depleted FNR (apo-FNR) suggested that removal of FAD from holo-FNR produced an intermediate conformational state with partially disrupted secondary and tertiary structures. Small angle x-ray scattering indicated that apo-FNR assumes a conformation that is less globular in comparison with holo-FNR but is not completely chain-like. Interestingly, the replacement of tyrosine 95 responsible for FAD binding with alanine resulted in a molecular form similar to apo-protein of the wild-type enzyme. Both apo- and Y95A-FNR species bound to Cibacron Blue affinity resin, indicating the presence of a native-like conformation for the NADP(+) binding domain. On the other hand, no evidence was found for the existence of folded conformations in the FAD binding domains of these proteins. These results suggested that FAD-depleted FNR assumes a partially folded structure with a residual NADP(+) binding domain but a disordered FAD binding domain.     

PAS domain of the Aer redox sensor requires C-terminal residues for native-fold formation and flavin adenine dinucleotide binding

The Aer protein in Escherichia coli is a membrane-bound, FAD-containing aerotaxis and energy sensor that putatively monitors the redox state of the electron transport system. Binding of FAD to Aer requires the N-terminal PAS domain and residues in the F1 region and C-terminal HAMP domain. The PAS domains of other PAS proteins are soluble in water. To investigate properties of the PAS domain, we subcloned segments of the aer gene from E. coli that encode the PAS domain with and without His6 tags and expressed the PAS peptides in E. coli. The 20-kDa His6-Aer2-166 PAS-F1 fragment was purified as an 800-kDa complex by gel filtration chromatography, and the associating protein was identified by N-terminal sequencing as the chaperone protein GroEL. None of the N-terminal fragments of Aer found in the soluble fraction was released from GroEL, suggesting that these peptides do not fold correctly in an aqueous environment and require a motif external to the PAS domain for proper folding. Consistent with this model, peptide fragments that included the membrane binding region and part (Aer2-231) or all (Aer2-285) of the HAMP domain inserted into the membrane, indicating that they were released by GroEL. Aer2-285, but not Aer2-231, bound FAD, confirming the requirement for the HAMP domain in stabilizing FAD binding. The results raise an interesting possibility that residues outside the PAS domain that are required for FAD binding are essential for formation of the PAS native fold.     

Interaction of phosphorylase kinase from rabbit skeletal muscle with flavin adenine dinucleotide

The interaction of flavin adenine dinucleotide (FAD) with rabbit skeletal muscle phosphorylase kinase has been studied. Direct evidence of binding of phosphorylase kinase with FAD has been obtained using analytical ultracentrifugation. It has been shown that FAD prevents the formation of the enzyme-glycogen complex, but exerts practically no effect on the phosphorylase kinase activity. The dependence of the relative rate of phosphorylase kinase-glycogen complex formation on the concentration of FAD has cooperative character (the Hill coefficient is 1.3). Under crowding conditions in the presence of 1 M trimethylamine-N-oxide (TMAO), FAD has an inhibitory effect on self-association of phosphorylase kinase. The data suggest that the complex of glycogen metabolism enzymes in protein-glycogen particles may function as a flavin depot in skeletal muscle. Published in Russian in Biokhimiya, 2006, Vol. 71, No. 6, pp. 808–814.     

NADPH-cytochrome P-450 oxidoreductase: flavin mononucleotide and flavin adenine dinucleotide domains evolved from different flavoproteins

The FMN-binding domain of NADPH-cytochrome P-450 oxidoreductase, residues 77-228, is homologous with bacterial flavodoxins, while the FAD-binding domain, residues 267-678, shows a high degree of similarity to two FAD-containing proteins, ferredoxin-NADP+ reductase and NADH-cytochrome b5 reductase. Comparison of these proteins to glutathione reductase, a flavoprotein whose three-dimensional structure is known, has permitted tentative identification of FAD- and cofactor-binding residues in these proteins. The remarkable conservation of sequence between NADPH-cytochrome P-450 oxidoreductase and ferredoxin-NADP+ reductase, coupled with the homology of the FMN-binding domain of the oxidoreductase with the bacterial flavodoxins, implies that NADPH-cytochrome P-450 oxidoreductase arose as a result of fusion of the ancestral genes for these two functionally linked flavoproteins.     

Interfacial behavior of uracil derivatives

The adsorption of a number of methylated uracil derivatives and of 5-fluorouracil has been studied by surface electrochemical methods at a mercury electrode. All derivatives exhibit an initial or dilute adsorption region where they are adsorbed flat on the electrode surface and are bound by pi-electron overlap with the electrode. Uracil, thymine, 1,5-dimethyl-uracil, 5,6-dimethyluracil, 1,5,6-trimethyluracil and 5-fluorouracil undergo a surface reorientation from the initial flat solution activities for each compound. An unsubstituted N(3)-H group is an absolute requirement for a uracil derivative to be capable of adopting the perpendicular surface stance. In the perpendicular orientation the uracil derivative appears to be bound to the electrode primarily via a N(3)-H--(-) electrode bond although a similar but weaker hydrogen bond can be formed via the N(1)-H group for certain compounds.     

Redox properties of the isolated flavin mononucleotide- and flavin adenine dinucleotide-binding domains of neuronal nitric oxide synthase

Electron transfer through neuronal nitric oxide synthase (nNOS) is regulated by the reversible binding of calmodulin (CaM) to the reductase domain of the enzyme, the conformation of which has been shown to be dependent on the presence of substrate, NADPH. Here we report the preparation of the isolated flavin mononucleotide (FMN)-binding domain of nNOS with bound CaM and the electrochemical analysis of this and the isolated flavin adenine dinucleotide (FAD)-binding domain in the presence and absence of NADP(+) and ADP (an inhibitor). The FMN-binding domain was found to be stable only in the presence of bound CaM/Ca(2+), removal of which resulted in precipitation of the protein. The FMN formed a kinetically stabilized blue semiquinone with an oxidized/semiquinone reduction potential of -179 mV. This is 80 mV more negative than the potential of the FMN in the isolated reductase domain, that is, in the presence of the FAD-binding domain. The FMN semiquinone/hydroquinone redox couple was found to be similar in both constructs. The isolated FAD-binding domain, generated by controlled proteolysis of the reductase domain, was found to have similar FAD reduction potentials to the isolated reductase domain. Both formed a FAD-hydroquinone/NADP(+) charge-transfer complex with a long-wavelength absorption band centered at 780 nm. Formation of this complex resulted in thermodynamic destabilization of the FAD semiquinone relative to the hydroquinone and a 30 mV increase in the FAD semiquinone/hydroquinone reduction potential. Binding of ADP, however, had little effect. The possible role of the nicotinamide/FADH(2) stacking interaction in controlling electron transfer and its likely dependence on protein conformation are discussed.     

The Aer protein of Escherichia coli forms a homodimer independent of the signaling domain and flavin adenine dinucleotide binding

In vivo cross-linking between native cysteines in the Aer receptor of Escherichia coli showed dimer formation at the membrane anchor and in the putative HAMP domain. Dimers also formed in mutants that did not bind flavin adenine dinucleotide and in truncated peptides without a signaling domain and part of the HAMP domain.     

Free radicals in the flavin reduction reaction. Interaction of reduced nicotinamide adenine dinucleotide with flavin in solution]

The study of the flavin mononucleotide (FMN)-reduced nicotinamide adeninedinucleotide (NADH) reaction was carried out both under aerobic and anaerobic conditions, using spectrophotometric and titrimetric methods. The consumption of NADH was shown to exceed two times the consumption of FMN in the anaerobic reaction and the rate constant in the aerobic reaction was found to be about 4 times of that of the anaerobic reaction. Moreover, the replacement of anaerobic conditions by aerobic ones at pH 5.0 resulted in a considerable increase of proton consumption rate in the reaction course. The data obtained are contradictory to the generally accepted hypothesis of hydrid-ion transfer in the reaction of NADH oxidation. It was assumed that this reaction proceeded through a homolytic pathway.