flavin-adenine-dinucleotide and imidazole

AppA, a transcriptional antirepressor, regulates the steady expression of photosynthesis genes in Rhodobacter sphaeroides in response to high-intensity blue light and to redox signals. Its blue-light sensing is mediated by an N-terminal BLUF domain, a member of a novel flavin fold. The photocycle of this domain (AppA(5-125)) includes formation of a slightly red-shifted long-lived signaling state, which is formed directly from the singlet excited state of the flavin on a subnanosecond time scale [Gauden et al. (2005) Biochemistry 44, 3653-3662]. The red shift of the absorption spectrum of this signaling state has been attributed to a rearrangement of its hydrogen-bonding interactions with the surrounding apoprotein. In this study we have characterized an AppA mutant with an altered aromatic amino acid: W104F. This mutant exhibits an increased lifetime of the singlet excited state of the flavin chromophore. Most strikingly, however, it shows a 1.5-fold increase in its quantum yield of signaling state formation. In addition, it shows a slightly increased rate of ground-state recovery. On top of this, the presence of imidazole, both in this mutant protein and in the wild-type BLUF domain, significantly accelerates the rate of ground-state recovery, suggesting that this rate is limited by rearrangement of (a) hydrogen bond(s). In total, an approximately 700-fold increase in recovery rate has been obtained, which makes the W104F BLUF domain of AppA, for example, suitable for future analyses with step-scan FTIR. The rate of ground-state recovery of the BLUF domain of AppA follows Arrhenius kinetics. This suggests that this domain itself does not undergo large structural changes upon illumination and that the structural transitions in full-length AppA are dominated by interdomain rearrangements.

Topics: Amino Acids, Aromatic; Bacterial Proteins; Base Sequence; Flavin-Adenine Dinucleotide; Flavoproteins; Hydrogen Bonding; Imidazoles; Light; Molecular Structure; Photosynthesis; Protein Structure, Tertiary; Repressor Proteins; Spectroscopy, Fourier Transform Infrared; Thermodynamics; Transcription Factors

The flavoprotein nitroalkane oxidase catalyzes the oxidation of primary and secondary nitroalkanes to aldehydes and ketones, respectively, transferring electrons to oxygen to form hydrogen peroxide. The steady-state kinetic mechanism of the active flavin adenine dinucleotide-(FAD-) containing form of the enzyme has been determined with nitroethane at pH 7 to be bi-ter ping-pong, with oxygen reacting with the free reduced enzyme after release of the aldehyde product. The V(max) value is 5.5 +/- 0.3 s(-)(1) and the K(m) values for nitroethane and oxygen are 3.3 +/- 0.6 and 0.023 +/- 0.007 mM, respectively. The free reduced enzyme forms a dead-end complex with nitroethane, with a K(ai) value of 30 +/- 6 mM. Acetaldehyde and butyraldehyde are noncompetitive inhibitors versus nitroethane due to formation of a dead-end complex between the oxidized enzyme and the product. Acetaldehyde is an uncompetitive inhibitor versus oxygen, indicating that an irreversible isomerization of the free reduced enzyme occurs before the reaction with oxygen. Addition of unprotonated imidazole results in a 5-fold increase in the V(max) value, while the V/K values for nitroethane and oxygen are unaffected. A 5-fold increase in the K(ai) value for nitroethane and a 6.5-fold increase in the K(ii) value for butyraldehyde are observed in the presence of imidazole. These results are consistent with the isomerization of the free reduced enzyme being about 80% rate-limiting for catalysis and with a model in which unprotonated imidazole accelerates the rate of isomerization.

Topics: Acetaldehyde; Binding, Competitive; Dioxygenases; Enzyme Activation; Enzyme Inhibitors; Ethane; Flavin-Adenine Dinucleotide; Fusarium; Imidazoles; Kinetics; Nitrites; Nitroparaffins; Oxygenases; Structure-Activity Relationship; Substrate Specificity

Benziman, Moshe (The Hebrew University of Jerusalem, Jerusalem, Israel), and Y. Galanter. Flavine adenine dinucleotide-linked malic dehydrogenase from Acetobacter xylinum. J. Bacteriol. 88:1010-1018. 1964.-The properties of the pyridine nucleotide-nonlinked malic dehydrogenase of Acetobacter xylinum were investigated in the supernatant fluid obtained by high-speed centrifugation of sonic extracts. Ferricyanide, phenazine methosulfate, and to a lesser extent dichlorophenolindophenol were active as oxidants for malate oxidation. After acid ammonium sulfate precipitation, the enzyme lost its malate-oxidizing activity. The enzyme was reactivated by low concentrations of flavine adenine dinucleotide (FAD) but not by flavine mononucleotide (FMN) or riboflavine. Atabrine inhibited the enzyme, and the inhibition was relieved by FAD but not by FMN or riboflavine. Malate-oxidizing activity was inhibited by hematin. The inhibition was prevented by imidazole or globin. o-Phenanthroline, 8-hydroxy quinoline, alpha,alpha'-dipyridyl, and p-chloromercuribenzoate inhibited malate oxidation. Amytal markedly inhibited oxidation of malate in the presence of oxygen, phenazine methosulfate, or dichlorophenolindophenol, but not in the presence of ferricyanide. The results suggest that the malic dehydrogenase of A. xylinum is a FAD enzyme, which contains an ironbinding site essential for its activity. Nonheme iron and sulfhydro groups are possibly involved in enzyme activity. The malic dehydrogenase is functionally linked to the cytochrome chain.

Topics: Acetobacter; Adenine; Amobarbital; Enzyme Inhibitors; Flavin Mononucleotide; Flavin-Adenine Dinucleotide; Gluconacetobacter xylinus; Heme; Imidazoles; Iron; Israel; Malate Dehydrogenase; Malates; Pharmacology; Phenanthrolines; Phenazines; Phenols; Quinacrine; Quinolines; Research; Riboflavin