Kinetic mechanism and quaternary structure of Aminobacter aminovorans NADH:Flavin oxidoreductase: An unusual flavin reductase with bound flavin. Aminobacter aminovorans NADH:Flavin oxidoreductase His140: A highly conserved residue critical for NADH bind

Flavin oxidoreductases (flavin reductases) are enzymes that exploit NADH, NADPH, or both with similar efficiencies to reduce the natural flavins: riboflavin, FMN, and FAD.; The objective of this dissertation was to investigate homodimeric FRD Aa as a model NADH-specific enzyme in order to compare and contrast it with other well-characterized flavin reductases. Evidence from this work favors the classification of FRDAa, as a flavin cofactor utilizing enzyme. The isolated native FRDAa contained about 0.5 bound FMN per enzyme monomer, but one bound flavin cofactor per monomer was obtainable in the presence of excess FMN or riboflavin. In addition, FRDAa holoenzyme also utilized FMN, riboflavin, or FAD as a substrate. Steady-state kinetic results indicated an ordered sequential mechanism with NADH as the first binding substrate and reduced FMN as the first leaving product. The FMN bound to the native FRDAa can be fully reduced by NADH and subsequently reoxidized by oxygen. No binding of NADH to the apoenzyme was detected.; For other flavin reductases, it has been found that a single residue is often responsible for pyridine nucleotide specificity. In the case of FRD Aa a sequence alignment with several known and hypothetical flavoproteins in the same subfamily reveals within the flavin reductase active site domain a conserved GDH motif, which is believed to be responsible for the enzyme and NADH interaction. Mutation of the His140 in this GDH motif to alanine reduced FRDAa activity to <3%. Similar to the wild type enzyme, the mutant is able to bind one FMN per monomer under saturating conditions, although with slightly weakened affinity. Also like the native enzyme, H140A FRDAa was able to reduce the FMN cofactor by NADH although much less efficiently. Kinetic analysis indicated that the His140 residue of FRD Aa was essential to NADH binding as well as important for the reduction of the FMN cofactor. For the native enzyme, the cofactor reduction was followed by at least one slower step in the catalytic pathway. (Abstract shortened by UMI.)...