Comparing the temperature dependence of FMN to heme electron transfer in full length and truncated inducible nitric oxide synthase proteins.

The FMN-heme interdomain (intraprotein) electron transfer (IET) kinetics in full length and oxygenase/FMN (oxyFMN) construct of human iNOS were determined by laser flash photolysis over the temperature range from 283 to 304K. An appreciable increase in the rate constant value was observed with an increase in the temperature. Our previous viscosity study indicated that the IET process is conformationally gated, and Eyring equation was thus used to analyze the temperature dependence data. The obtained magnitude of activation entropy for the IET in the oxyFMN construct is only one-fifth of that for the holoenzyme. This indicates that the FMN domain in the holoenzyme needs to sample more conformations before the IET takes place, and that the FMN domain in the oxyFMN construct is better poised for efficient IET.

Identification of a flavin mononucleotide module residue critical for activity of inducible nitrite oxide synthase.

Inducible NO synthase (iNOS) contains an amino-terminal oxygenase domain, a carboxy-terminal reductase domain, and an intervening calmodulin-binding domain. For the synthesis of NO, iNOS is active as a homodimer formed by oxygenase domains, while the reductase domain is required to transfer electrons from NADPH. In this study, we identify glutamate 658 in the FMN domain of human iNOS to be a critical residue for iNOS activity and we explore the underlying mechanism for such role. Mutation of glutamate to aspartate almost abolished iNOS activity and reduced dimer formation. Substitution of this residue with noncharged alanine and glutamine, or positively charged lysine did not affect dimer formation and maintained around 60% of iNOS activity. These results suggest that the negative charge specific to glutamate plays an important role in iNOS activity.

The terminal step in vitamin C biosynthesis in Trypanosoma cruzi is mediated by a FMN-dependent galactonolactone oxidase.

Humans lack the ability to synthesize vitamin C (ascorbate) due to the absence of gulonolactone oxidase, the last enzyme in the biosynthetic pathway in most other mammals. The corresponding oxidoreductase in trypanosomes therefore represents a target that may be therapeutically exploitable. This is reinforced by our observation that Trypanosoma cruzi, the causative agent of Chagas' disease, lacks the capacity to scavenge ascorbate from its environment and is therefore dependent on biosynthesis to maintain intracellular levels of this vitamin. Here, we show that T. cruzi galactonolactone oxidase (TcGAL) can utilize both L-galactono-gamma-lactone and D-arabinono-gamma-lactone as substrates for synthesis of vitamin C, in reactions that obey Michaelis-Menten kinetics. It is >20-fold more active than the analogous enzyme from the African trypanosome Trypanosoma brucei. FMN is an essential cofactor for enzyme activity and binds to TcGAL non-covalently. In other flavoproteins, a histidine residue located within the N-terminal flavin-binding motif has been shown to be crucial for cofactor binding. Using site-directed mutagenesis, we show that the corresponding residue in TcGAL (Lys-55) is not essential for this interaction. In contrast, we find that histidine and tryptophan residues (His-447 and Trp-448), localized within a C-terminal motif (HWXK) that is a feature of ascorbate-synthesizing enzymes, are necessary for the FMN association. The conserved lysine residue within this motif (Lys-450) is not required for cofactor binding, but its replacement by glycine renders the protein completely inactive.

Mitochondrial Complex I: structure, function, and implications in neurodegeneration.

Mitochondrial Complex I (NADH Coenzyme Q oxidoreductase) is the least understood of respiratory complexes. In this review we emphasize some novel findings on this enzyme that are of relevance to the pathogenesis of neurodegenerative diseases. Besides Coenzyme Q (CoQ), also oxygen may be an electron acceptor from the enzyme, with generation of superoxide radical in the mitochondrial matrix. The site of superoxide generation is debated: we present evidence based on the rational use of several inhibitors that the one-electron donor to oxygen is an iron-sulphur cluster, presumably N2. On this assumption we present a novel mechanism of electron transfer to the acceptor, CoQ. Strong evidence is accumulating that electron transfer from Complex I to Complex III via CoQ is not performed by operation of the CoQ pool but by direct channelling within a super-complex including Complex I, Complex III and bound CoQ. Besides structural evidence of a Complex I -Complex III aggregate obtained by native electrophoresis, we have obtained kinetic evidence based on metabolic flux analysis, demonstrating that Complexes I and III behave as an individual enzyme. Quantitative and qualitative changes of phospholipids, including peroxidation, may affect the supercomplex formation. Complex I is deeply involved in pathological changes, including neurodegeneration. Maternally inherited mutations in mitochondrial DNA genes encoding for Complex I subunits are at the basis of Leber's Hereditary Optic Neuropathy; a decrease of electron transfer in the complex, due to the mutations, is not sufficient per se to explain the clinical phenotype, and other factors including proton translocation and oxygen radical generation have been considered of importance. Complex I changes are also involved in more common neurological diseases of the adult and old ages. In this review we discuss Parkinson's disease, where the pathogenic involvement of Complex I is better understood; the accumulated evidence on the mode of action of Complex I inhibitors and their effect on oxygen radical generation is discussed in terms of the aetiology and pathogenesis of the disease.

Staphylococcus aureus NfrA (SA0367) is a flavin mononucleotide-dependent NADPH oxidase involved in oxidative stress response.

The NfrA protein, a putative essential oxidoreductase in the soil bacterium Bacillus subtilis, is induced under heat shock and oxidative stress conditions. In order to characterize the function of an homologous NfrA protein in Staphylococcus aureus, an nfrA deletion strain was constructed, the protein was purified, the enzymatic activity was determined, and the transcriptional regulation was investigated. The experiments revealed that NfrA is not essential in S. aureus. The purified protein oxidized NADPH but not NADH, producing NADP in the presence of flavin mononucleotide, suggesting that NfrA is an NADPH oxidase in S. aureus. In addition, the NfrA enzyme showed nitroreductase activity and weak disulfide reductase activity. Transcription was strongly induced by ethanol, diamide, and nitrofurantoin. Hydrogen peroxide induced nfrA transcription only at high concentrations. The expression of nfrA was independent of the alternative sigma factor sigma(B). Furthermore, the transcriptional start site was determined, which allowed identification of a PerR box homologous sequence upstream of the nfrA promoter. The observations presented here suggest that NfrA is a nonessential NADPH oxidoreductase which may play a role in the oxidative stress response of S. aureus, especially in keeping thiol-disulfide stress in balance.

Working at the cutting edge: the creation of allosteric ribozymes.

The appropriate folding of catalytic RNA is a prerequisite for effective catalysis. A novel ribozyme, the maxizyme, has been generated and its activity can be controlled allosterically. The maxizymes work both in vitro and in vivo indicating the potential utility of this novel class of ribozyme as a gene-inactivating agent with a biosensor function.

Lumiflavin and lumichrome transport in the central nervous system.

The transport of the lipid-soluble sugarless flavins, [14C]lumiflavin and [14C]lumichrome, into an from the isolated choroid plexus and brain slices was studied in vitro. The isolated choroid plexus accumulated both [14C]flavins by a saturable, energy-requiring process that did not depend on binding or intracellular metabolism of the [14C]flavins. Both sugar-containing and sugarless flavins, as well as cyclic organic acids, significantly inhibited [14C]lumiflavin and [14C]lumichrome uptake by the isolated choroid plexus. Within 2.5 min, 75% of the [14C]lumiflavin accumulated by the isolated choroid plexus was released into the medium. Brain slices accumulated [14C]lumiflavin by a saturable process that did not meet all the criteria for active transport. Ninety-five percent of the [14C]lumiflavin accumulated by brain slices was released into the medium within 7.5 min. In vivo, 2 h after the intraventricular injection of 6.5 nmol [14C]lumiflavin, almost all of the [14C]flavin was cleared from the CNS. Addition of 3.5 mumol FMN to the intraventricular injectate significantly decreased the clearance of [14C]lumiflavin from the CNS. These studies document that the sugarless flavins are transported by the flavin transport systems in the CNS.