Flavins cofactor function

The small subunit is composed of two domains. The N-terminal domain shows the characteristic architecture of flavodoxin with the phosphate moiety of the flavin cofactor occupying the binding pocket of the proximal [4Fe-4S] cluster. This N-terminal domain, including the proximal cluster, is found in all [NiFe] hydrogenases and is consequently an essential feature, both structural and functional, of these enzymes. By contrast, the C-terminal domain that binds the other [FeS] clusters is less organised and more variable in [FeS] cluster content and amino acid sequence. 

Metalloenzymes or metal ion-activated enzymes catalyze an enormous variety of organic reactions that are not restricted to any particular reaction class, but appear as catalysts for all types of reactions. Thus neither the presence of the metal ion nor the reaction type seems to be restrictive as far as metal-assisted enzyme catalysis is concerned. In some cases the metal ion appears to function as an electron acceptor or donor, but flavin cofactors have substituted as redox centers during evolution in some enzymes. 

A model of a flavin-based redox enzyme was prepared.[15] Redox enzymes are often flavoproteins containing flavin cofactors flavin adenine dinucleotide (FAD) or flavin mononucleotide (FMN). They mediate one- or two-electron redox processes at potentials which vary in a range of more than 500 mV. The redox properties of the flavin part must be therefore tuned by the apoenzyme to ensure the specific function of the enzyme. Influence by hydrogen bonding, aromatic stacking, dipole interactions and steric effects have been so far observed in biological systems, but coordination to metal site has never been found before. Nevertheless, the importance of such interactions for functions and structure of other biological molecules make this a conceivable scenario. 

In an attempt to mimic the function of these enzymes, PDC was derivatized with the flavin analogs 8-bromoacetyl- and 6-bromoacetyl-10-methylisoalloxazine (the heterocyclic core of flavin cofactors shown in equation 20 below). The former led to total... 

Flavin-dependent monooxygenases function through a reaction cycle that is schematically presented in Fig. 4.79. These enzymes perform their catalytic reaction through the generation of a reactive form of the flavin cofactor, formed by 2-electron reduction of the flavin followed by its reaction with molecular oxygen. As a result, the so-called C(4a)-peroxyflavin intermediate is formed, whose electrophilic reactivity is further increased by protonation of the distal oxygen of the peroxide to yield the C(4a)-hydroperoxyflavin form of the cofactor (Fig. 4.80). 

Many enzymes use the flavin cofactor at the active site the fluorescent active site approach can be applied to study these enzymes at the single-molecule level. Other naturally fluorescent enzymes, like those that contain NAD cofactors, can in principle be studied, although the bluer fluorescence of NAD poses a technical challenge for singlemolecule fluorescence detection. As the approach uses the natural fluorescence of the enzyme, no labeling with fluorescent probes is needed, offering no or minimum perturbation on the enzyme structure and function. 

In a manner similar to the structures of the nicotinamide cofactors, the flavin cofactors FMN/FMNH2 and FAD/FADH2 also have structures that resolve into a chemically reactive unit, the flavin nucleus, and a large ancillary structure that has the function of binding the cofactor in a specific orientation to the host enzyme, as emerges from Fig. 4.2. Here again the flavin nucleus and thus the strictly chemical properties are common to the two coenzymes, which differ in the ancillary part of the structure. 

Treatment of the blue form of the lyase with dithionite or irradiation at wavelengths greater than 520 nm in the presence of DTT produces fully reduced flavin cofactor (FADH2) and results in a dramatic increase in activity and quantum yield (146, 153, 154). This suggests that the catalytically active oxidation state of the cofactor may be the reduced flavin, and that it is this form of the cofactor that functions as an electron donor in catalysis (155). Subsequent studies by Sancar and co-workers indicate that E. coli DNA-PL does not contain the flavin semiquinone radical in vivo, and that the blue, radical enzyme does not catalyze dimer cleavage (156). 

Redox-active cofactors are important species in biological systems, playing vital roles in redox and electron-transfer processes. Among the structurally and functionally diverse redox enzymes, flavoproteins containing the flavin cofactors flavin adenine dinucleotide (FAD) or flavin mononucleotide (FMN) are involved in many different biochemical processes serving as a highly versatile redox... 

Functional interactions in cytochrome P450BM3. Evidenee that NADP(H) binding controls redox potentials of the flavin cofactors. Biochemistry 39, 12699-12707. 

Fig. 13.1.2. Schematic representation of the structure and function of Compiex i. The compiex can be resoived into three sub-compiexes lo, (a subset of lo) and /l, arranged as indicated [28]. The NADH binding site, flavin cofactor (FMN) and all of the know iron sulfur centers (Fe) are carried...

Some FeNO complexes have been synthesized to mimic the structure and function of nitric oxide reductase enzymes, which can be separated into two classes. One class utilizes a heme/nonheme active site to reduce two equivalents of NO into N2O and is found in denitrifying bacteria (NorBC) (18,19). Another class is found in pathogenic bacteria such as Helicobacter pylori. Neisseria meningitides, and Salmonella enterica. These microbes have evolved ways to handle attack by NO by converting it to relatively ben p N2O through the expression of nitric oxide reductases utilizing a nonheme diiron protein that has a flavin cofactor within 4 A of the active site metals (FNORs Figure 7) (16,17). A few recent (2011—2014) FeNO complexes have been constructed to model FNORs in order to probe the mechanism of this di-Fe enzyme. These complexes will be discussed below. 

The ability of flavins to engage in both 1-electron and 2-electron redox chemistry is key to their functions in electron transfer. In POR, they are an essential intermediate between NADPH, a two-electron donor, and the heme of P450, a one-electron acceptor. Furthermore, utihzation of two flavins, located in separate domains, provides a mechanism for control of the kinetics of electron transfer by regulating the distance between, and the relative orientation of, the two flavins. The flavin cofactors can exist as the oxidized (ox), one-electron reduced semiquinone (sq), and two-electron, fully reduced (red) forms (Fig. 2.3). 

The functional diversity of flavoproteins results from the broad range of redox potentials that are accessible to the flavin cofactors, as well as their ability to switch between one or two electron redox chemistry. In solution, flavins are found in equilibrium between the oxidized, reduced and the semi-quinone radical forms, and have a redox potential of about —210 mV (versus the normal hydrogen electrode) at neutral pH. However, in the protein-bound form, the redox equilibrium can be shifted and the redox potential may span up to 600 mV (Massey 2000). This arises from the fact that flavin-protein interactions may engage a number of non-covalent interactions such as 7i-stacking, hydrophobic effects, hydrogen bonding and electrostatic interactions, which will ultimately determine the flavin redox potential. 

Enzymes containing amino acid radicals are generally associated with transition metal ions—typically of iron, manganese, cobalt, or copper. In some instances, the metal is absent it is apparently replaced by redox-active organic cofactors such as S -adenosylmethionine or flavins. Functionally, their role is analogous to that of the metal ion in metalloproteins. 

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