Flavin catalysis

Bruice, T.C. Models and flavin catalysis. In Progress in bioorganic chemistry, Vol. 4, p. 1, E.T. Kaiser, F. J. Kezdy, (eds.), New York-London-Sidney-Toronto Wiley 1976... 

Bniice, T. C. Some physical organic studies dealing with flavin catalysis. In Flavin and flavoproteins (Singer, T. P. ed.) pp. 244-260. Amsterdam, Elsevier 1976... 

Flavin catalysis may involve three oxidation levels of the ring, and each of these may exist as a cation, anion or neutral compound, giving a total of 9 forms. The oxidation levels are flavoquinone (oxidized) flavosemiquinone (half-reduced) flavohydroqui-none (reduced). Some catalytic mechanisms involve only one electron, i.e. the flavoquinone is converted to the semiquinone, or the semiquinone is converted to the hydroquinone. Other mechanisms involve two electrons, and the flavin shuttles back and forth between the quinone and the hydroquinone states. FAD is biosynthesized from flavin mononucleotide by the action of FAD pyrophosphatase (EC 3.6.1.8) FMN + ATPseFAD + PP,. 

Studies of addition of thiolates to flavans showing that addition to a 4a- or S-position of an isoalloxazine ring may be implicated in flavin catalysis of thiol-disulphide oxidation, are sufficiently broad to include dithiols and arenesulphinic acids. ... 

Catalysis by flavoenzymes has been reviewed and various analogues of FAD have been prepared e.g. P -adenosine-P -riboflavin triphosphate and flavin-nicotinamide dinucleotide ) which show little enzymic activity. The kinetic constants of the interaction between nicotinamide-4-methyl-5-acetylimidazole dinucleotide (39) and lactic dehydrogenase suggest the presence of an anionic group near the adenine residue at the coenzyme binding site of the enzyme. ... 

Flavins — Riboflavin is first of all essential as a vitamin for humans and animals. FAD and FMN are coenzymes for more than 150 enzymes. Most of them catalyze redox processes involving transfers of one or two electrons. In addition to these well known and documented functions, FAD is a co-factor of photolyases, enzymes that repair UV-induced lesions of DNA, acting as photoreactivating enzymes that use the blue light as an energy source to initiate the reaction. The active form of FAD in photolyases is their two-electron reduced form, and it is essential for binding to DNA and for catalysis. Photolyases contain a second co-factor, either 8-hydroxy-7,8-didemethyl-5-deazariboflavin or methenyltetrahydrofolate. ... 

All characterized BVMOs contain a flavin cofactor that is crucial for catalysis while NADH or NADPH is needed as electron donor. An interesting observation is the fact that most reported BVMOs are soluble proteins. This is in contrast to many other monooxygenase systems that often are found to be membrane-bound or membrane-associated. In 1997, Willetts concluded from careful inspection of... 

Pollock RJ, LB Hersh (1973) A-methylglutamate synthetase. The use of flavin mononucleotide in oxidative catalysis. J Biol Chem 248 6724-6733. 

We next focus on the use of fixed-site cofactors and coenzymes. We note that much of this coenzyme chemistry is now linked to very local two-electron chemistry (H, CH3", CH3CO-, -NH2,0 transfer) in enzymes. Additionally, one-electron changes of coenzymes, quinones, flavins and metal ions especially in membranes are used very much in very fast intermediates of twice the one-electron switches over considerable electron transfer distances. At certain points, the chains of catalysis revert to a two-electron reaction (see Figure 5.2), and the whole complex linkage of diffusion and carriers is part of energy transduction (see also proton transfer and Williams in Further Reading). There is a variety of additional coenzymes which are fixed and which we believe came later in evolution, and there are the very important metal ion cofactors which are separately considered below. 

While cytochrome P-450 catalyzes the interaction with substrates, a final step of microsomal enzymatic system, flavoprotein NADPH-cytochrome P-450 reductase catalyzes the electron transfer from NADPH to cytochrome P-450. As is seen from Reaction (1), this enzyme contains one molecule of each of FMN and FAD. It has been suggested [4] that these flavins play different roles in catalysis FAD reacts with NADPH while FMN mediates electron... 

The addition of cofactors to antibodies is a sure means to confer a catalytic activity to them insofar as this cofactor is responsible for the activity. Indeed for many enzymes, the interaction with cofactors such as thiamins, flavins, pyridoxal phosphate, and ions or metal complexes is absolutely essential for the catalysis. It is thus a question there of building a new biocatalyst with two partners the cofactor responsible for the catalytic activity, and the antibody which binds not only the cofactor but also the substrate that it positions in a specific way one with respect to the other, and can possibly take part in the catalysis thanks to some of its amino acids. 

Flavin mononucleotide, 3absorption coefficients, 36 270 active site, 36 265-267 catalysis and electron transfer, 36 275-287 carbanion mechanism, 36 277-282 electron acceptors, 36 285-287 electron transfer pathway, 36 275-276, 282-285... 

NADPH oxidation and NO synthesis by the enzyme, it supports a role for reduction of the heme iron in catalysis, and may explain why NOS functions only as an NADPH-dependent reductase in the absence of bound calmodulin (Klatt et ai, 1993). The mechanism of calmodulin gating is envisioned to involve a conformational change between the reductase and oxygenase domains of NOS, such that an electron transfer between the terminal flavin and heme iron becomes possible. Calmodulin may also have a distinct role within the NOS reductase domain, in that its binding dramatically increases reductase activity of the enzyme toward cytochrome c (Klatt et al., 1993 Heinzel et al., 1992). However, it is clear that several other NOS functions occur independent of calmodulin, including the binding of L-arginine and NADPH, and transfer of NADPH-derived electrons into the flavins (Abu-Soud and Stuehr, 1993). 

Flavohydroquinone bound to apoproteins plays a very important role in flavo-protein-catalysis, either in the electron-transfer to substrates or other enzymes or in the oxygen activation reaction. The chemical reactivity of 1,5-dihydroflavin bound to apoproteins can differ drastically from that of free flavin. The reactivity is likely governed by factors such as the conformation of the bound flavohydroquinone and the ionization state (cf. below). 

Pyridine nucleotide-dependent flavoenzyme catalyzed reactions are known for the external monooxygenase and the disulfide oxidoreductases However, no evidence for the direct participation of the flavin semiquinone as an intermediate in catalysis has been found in these systems. In contrast, flavin semiquinones are necessary intermediates in those pyridine nucleotide-dependent enzymes in which electron transfer from the flavin involves an obligate 1-electron acceptor such as a heme or an iron-sulfur center. Examples of such enzymes include NADPH-cytochrome P4S0 reductase, NADH-cytochrome bs reductase, ferredoxin — NADP reductase, adrenodoxin reductase as well as more complex enzymes such as the mitochondrial NADH dehydrogenase and xanthine dehydrogenase. 

Both stopped-flow and rapid freeze quench kinetic techniques show that the substrate reduces the flavin to its hydroquinone form at a rate faster than catalytic turnover Reoxidation of the flavin hydroquinone by the oxidized Fe4/S4 center leads to formation of a unique spin-coupled species at a rate which appears to be rate limiting in catalysis. Formation of this requires the substrate since dithionite reduction leads to flavin hydroquinone formation and a rhombic ESR spectrum typical of a reduced iron-sulfur protein . The appearance of such a spin-coupled flavin-iron sulfur species suggests the close proximity of the two redox centers and provides a valuable system for the study of flavin-iron sulfur interactions. The publication of further studies of this interesting system is looked forward to with great anticipation. 

It should be obvious from the literature discussed in this article that progress in our understanding of the properties of flavin semiquinones and their role in flavoenzyme catalysis has increased dramatically over the past twenty years. This has been due to the application of sophisticated chemical and physical approaches, as well as to an increase in the number and diversity of flavoenzymes which have been purified to homogeneity in quantities sufficient for extensive study. 

Figure 14-2 (A) Stereoscopic view of the active site of pyruvate oxidase from the bacterium Lactobacillus plantarium showing the thiamin diphosphate as well as the flavin part of the bound FAD. The planar structure of the part of the intermediate enamine that arises from pyruvate is shown by dotted lines. Only some residues that may be important for catalysis are displayed G35 , S36 , E59 , H89 , F12T, Q122 , R264, F479, and E483. Courtesy of Georg E. Schulz.119 (B) Simplified view with some atoms labeled and some side chains omitted. The atoms of the hypothetical enamine that are formed from pyruvate, by decarboxylation, are shown in green.

However, these experiments may not have established a mechanism for natural flavoprotein catalysis because the properties of 5-deazaflavins resemble those of NAD+ more than of flavins.239 Their oxidation-reduction potentials are low, they do not form stable free radicals, and their reduced forms don t react readily with 02. Nevertheless, for an acyl-CoA dehydrogenase the rate of reaction of the deazaflavin is almost as fast as that of natural FAD.238 For these enzymes a hydride ion transfer from the (3 CH (reaction type D of Table 15-1) is made easy by removal of the a-H of the acyl-CoA to form an enolate anion intermediate. 

---