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.     

Covalent attachment of flavin adenine dinucleotide (FAD) and flavin mononucleotide (FMN) to enzymes: the current state of affairs.

The first identified covalent flavoprotein, a component of mammalian succinate dehydrogenase, was reported 42 years ago. Since that time, more than 20 covalent flavoenzymes have been described, each possessing one of five modes of FAD or FMN linkage to protein. Despite the early identification of covalent flavoproteins, the mechanisms of covalent bond formation and the roles of the covalent links are only recently being appreciated. The main focus of this review is, therefore, one of mechanism and function, in addition to surveying the types of linkage observed and the methods employed for their identification. Case studies are presented for a variety of covalent flavoenzymes, from which general findings are beginning to emerge.     

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.     

Modification of flavin adenine dinucleotide in alcohol oxidase of the yeast Hansenula polymorpha.

Alcohol oxidase, a major peroxisomal protein of methanol-utilizing yeasts, may possess two different forms of flavin adenine dinucleotide, classical FAD and so-called modified FAD (mFAD). Conversion of FAD into mFAD was observed both in purified preparations of the enzyme and in cells grown in batch and continuous culture. The relative amount of mFAD in the enzyme varied from 5 to 95%, depending on the growth or storage conditions. The presence of mFAD led to a slight decrease in Vmax and a significant (about one order) decrease in the Km of alcohol oxidase with respect to methanol. The kinetics of modification measured in purified preparations of the enzyme obeyed first-order kinetics (k = 0.78 h-1). The modification process was strongly inhibited by methanol, formaldehyde or hydroxylamine. Modification observed in continuous culture under steady state conditions depended on the dilution rate and could also be described as a spontaneous first-order reaction (kapp = 0.27 h-1). FAD modification could only be detected in alcohol oxidase and not in other yeast peroxisomal flavoenzymes, such as D-amino acid oxidase from Candida boidinii.     

Monodehydroascorbate reductase from cucumber is a flavin adenine dinucleotide enzyme

Monodehydroascorbate reductase (EC 1.6.5.4) was purified from cucumber fruit to a homogeneous state as judged by polyacrylamide gel electrophoresis. The cucumber monodehydroascorbate reductase was a monomer with a molecular weight of 47,000. It contained 1 mol of FAD/mol of enzyme which was reduced by NAD(P)H and reoxidized by monodehydroascorbate. The enzyme had an exposed thiol group whose blockage with thiol reagents inhibited the electron transfer from NAD(P)H to the enzyme FAD. Both NADH and NADPH served as electron donors with Km values of 4.6 and 23 microM, respectively, and Vmax of 200 mol of NADH and 150 mol of NADPH oxidized mol of enzyme-1 s-1. The Km for monodehydroascorbate was 1.4 microM. The amino acid composition of the enzyme is presented. In addition to monodehydroascorbate, the enzyme catalyzed the reduction of ferricyanide and 2,6-dichloroindophenol but showed little reactivity with calf liver cytochrome b5 and horse heart cytochrome c. The kinetic data suggested a ping-pong mechanism for the monodehydroascorbate reductase-catalyzed reaction. Cucumber monodehydroascorbate reductase occurs in soluble form and can be distinguished from NADPH dehydrogenase, NADH dehydrogenase, DT diaphorase, microsome-bound NADH-cytochrome b5 reductase, and NADPH-cytochrome c reductase by its molecular weight, amino acid composition, and specificity of electron acceptors and donors.     

Unusual folded conformation of nicotinamide adenine dinucleotide bound to flavin reductase P.

The 2.1 A resolution crystal structure of flavin reductase P with the inhibitor nicotinamide adenine dinucleotide (NAD) bound in the active site has been determined. NAD adopts a novel, folded conformation in which the nicotinamide and adenine rings stack in parallel with an inter-ring distance of 3.6 A. The pyrophosphate binds next to the flavin cofactor isoalloxazine, while the stacked nicotinamide/adenine moiety faces away from the flavin. The observed NAD conformation is quite different from the extended conformations observed in other enzyme/NAD(P) structures; however, it resembles the conformation proposed for NAD in solution. The flavin reductase P/NAD structure provides new information about the conformational diversity of NAD, which is important for understanding catalysis. This structure offers the first crystallographic evidence of a folded NAD with ring stacking, and it is the first enzyme structure containing an FMN cofactor interacting with NAD(P). Analysis of the structure suggests a possible dynamic mechanism underlying NADPH substrate specificity and product release that involves unfolding and folding of NADP(H).     

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.     

Oxidation of dihydronicotinamide adenine dinucleotide by a flavin derivative of polyethylenimine

A modified Polyethylenimine has been prepared that has riboflavin attached to it, as well as hydrophobic groups. The catalytic efficiency toward oxidation of6 dihydronicotinamide adenine dinucleotide (NADH) by this flavopolymer is more than 100-fold greater than observed with small-molecular riboflavin. The products of the reaction in aerobic solution have been established to be the oxidized nicotinamide NAD⁺ and hydrogen peroxide. The kinetics fit a Michaelis-Menten mechanism. Thus, the versatility of modified polyethylenimines as catalysts has been extended from hydrolyses and decarboxylations to oxidation-reduction reactions also.     

Oligomeric forms of plant acetolactate synthase depend on flavin adenine dinucleotide

Acetolactate synthase (ALS, EC 4.1.3.18) has been extracted and partially purified from etiolated barley shoots (Hordeum vulgare L.). Multiple forms of this enzyme were separated by gel filtration and/or anion-exchange chromatography using fast protein liquid chromatography. It could be demonstrated that these two species are in equilibrium, which strongly depends on the structural role of flavin adenine dinucleotide and pyruvate. With 50 micromolar of flavin adenine dinucleotide in the medium most of the ALS aggregates as a high molecular weight form (M_r = 440,000), while 50 millimolar pyruvate facilitates dissociation into the smaller form (M_r = 200,000). Data are presented to show that two enzymatically active forms are not isozymes but different oligomeric species or aggregates of the basic 58-kilodalton subunit of ALS. These different ALS species exhibit little difference in feedback inhibition by valine, leucine and isoleucine or in inhibition by the sulfonylurea herbicide chlorsulfuron. Both aggregation forms show a broad pH-optimum between 6.5 and 7. Furthermore, the affinity for pyruvate and the amount of directly-formed acetoin indicate similar properties of these separated ALS forms.