Flavin enzymes

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Chapter 3 Flavin Enzymes:Biological Functions, Structure and Interactions

3.1. Introduction

Flavin-containing enzymes are class of enzymes which contain the flavin isoalloxazine heterocyclic ring as a cofactor. This cofactor can either be flavin mononucleotide (FMN) or flavin adenine dinucleotie (FAD). They both are constituted from riboflavin: riboflavin 5'-phosphate for FMN and an additionally attached adenosine monophosphate for FAD. The nucleotide part of the flavin ring does not participate in any chemical transformations, but is important for recognition and binding of the cofactor to the particular enzyme {REFs?}. The chemical formula of the both cofactors is shown in Figure 3.1

Experimental techniques as X-ray crystallography, electronic absorption spectroscopy, circular dichroism, and kinetic studies have been extensively applied in studying the flavin-containing enzymes {REFs?}. Experimental results provide an excellent background for the application of computational methods which can provide further crucial insights into atomistic, electronic structural and dynamical properties of the flavoenzymes that can not be gained solely by experiment. Computational chemistry methods have been successfully applied to investigate flavin enzymes and in particular in revealing their mechanisms, the role of the flavin cofactor and the effect of the protein environment {Senn, 2009 #2559; Ridder, 2003 #1172; Mulholland, 2001 #1114}.

A large number of crystal structures of FMN and FAD containing flavoproteins are currently available in the protein data bank (PDB) {REF}. It is notable that around 1-3% of the genes in bacterial and eukaryotic genomes encode flavin-binding proteins. The flavoprotein class of enzymes performs a large variety of biochemical processes that are involved in important biological functions such as electron transfer, dehydrogenation of a diversity of metabolites, light emission, activation of oxygen for oxidation {Berg, 2002 #176}. Flavoenzymes can also catalyze unusual chemical reactions, such as halogenation at the 7th position of an aromatic substrate - tryptophan {Murphy, 2006 #1792} and oxidative deamination of biogenic amines {Edmondson, 2004 #1366}. The combination of structural analyses with experimental ones such as fast kinetics and other studies provides accurate information about the structure-function relationships in flavoenzymes.

The flavin cofactor izoalloxazine ring can exist in the following redox states: oxidized and reduced (one-electron-semiquinone and fully-two-electron-hydroquinone reduced form) (Figure 3.2) {Berg, 2002 #176}. The spectroscopic properties of the forms of the izoalloxazine ring were widely studied and used in revealing of the reaction mechanisms of flavoenzymes {Senda, 2009 #2471}.

3.2.Flavin binding site

The chemically interesting part of the flavin cofactor is its tricyclic isoalloxazine moiety (Figure.3.1). Each of the states (redox, ionic, or electronic) has specific electronic, spectral and chemical properties which can be influenced by the protein environment. The flavin isoalloxazine ring is amphipathic and is formed by fusion between the hydrophobic dimethylbenzene and the hydrophilic pyrimidine ring. The redox potential of the two-electron reduction of the flavin is approximately 2200 mV. However, this value varies greatly between different flavoenzymes (e.g. from 2400 mV to 160 mV 13,14). It is thought that the proximity of a positive charge increases the redox potential, whereas a negative charge or a hydrophobic environment lowers its value {Senda, 2009 #2471}.

Many flavoenzymes have a covalently bound FAD and the covalent bond is likely to play a role in increasing the oxidative potential of the flavin. The isoalloxazine ring may adopt conformations that differ from planarity (e.g. polyamine oxidase, cholesterol oxidase and trimethylamine dehydrogenase {REFs?}) however its ability to form hydrogen bonds is exhibited in all enzymes. Flavoenzymes can catalyze reactions through formation of a covalent intermediate formed by the substrate attack on the flavin ring. For example, such covalent catalysis is exhibited by nitroalkane oxidase, which performs the oxidative degradation of nitroalkanes to products such as nitrite, hydrogen peroxide, and a carbonyl compound {Senda, 2009 #2471}.

3.3. Redox states of flavins

The spectroscopic characteristics of the flavin redox forms have been used extensively in revealing reaction mechanisms of flavoenzymes {van Berkel WJ, 1999 #2470}. Oxidized forms of flavin cofactors show aspecific absorption near 450 nm. The oxidized isoalloxazine ring has two pKa values: ~0 for N1 and ~10 for N3, therefore under physiological conditions the oxidized isoalloxazine ring of FAD and FMN is usually in a neutral form. One electron reduction transforms the oxidized flavin to the semiquinone form. The semiquinone has a pKa value of ~8.3 for the N5 atom. The neutral protonated semiquinone is blue (λmax ~560 nm) and the anionic semiquinone is red (λmax 390-410 nm and ~480 nm). Both semiquinone forms are reported for various flavoenzymes. When the isoalloxazine ring is fully reduced to a hydroquinone, flavin becomes nearly colourless (it is still pale yellow). As the hydroquinone has a pKa value of ~6.6 for the N1 atom, the fully reduced flavin could exist in neutral or anionic forms under physiological conditions.

3.4. Redox-dependent conformational change of the isoalloxazine ring in free flavin

The isoalloxazine ring of free oxidized flavins in crystal structures has a planar conformation {Fritchie, 1975 #2560}, whereas the fully reduced flavin adopts a bent conformation along the N5-N10 axis {Werner, 1970 #2561}. The conformation of the isoalloxazine ring in crystal structures can be influenced by steric effects due to crystal packing. In order to provide insights into flavin conformations in solution computational studies have been performed. Electronic structure calculations confirmed that the oxidized isoalloxazine ring is planar; however, it bends along the N5- N10 axis after two-electron reduction. The hybridization of the N5 and N10 atoms in the fully reduced flavin were calculated to be intermediate between sp2 and sp3 type. Calculations of the semiquinone form of the isoalloxazine ring predict a conformation very near to planar {Zheng, 1996 #2565}. However, NMR experiments of free flavins in solution demonstrate that fully reduced flavin in solution contains an sp2-hybridized N10 atom, and that the N5 atom also exhibits a mainly sp2 character, indicating that the fully reduced flavin is planar in water {Moonen, 2002 #2566}. The energy barrier for the bent-planar transition of the reduced flavin was estimated by NMR as being <4.8 kcal/mol. A theoretical calculation also showed that the energy barrier for the bent-planar transition was only ~6.4 kcal/mol {Zheng, 1996 #2565}. Therefore the isoalloxazine ring conformation may be sensitive to environmental effects (such us interactions with the protein environment). QM/MM calculations which take the effects of the protein environment into account could be very useful in the investigation of flavin conformations.

3.5. Reactivity of flavins in the protein

The redox properties of the isoalloxazine ring provide the ability for FAD and FMN to act as redox-active cofactors in flavoproteins that catalyze a diverse range of reactions. Isoalloxazine ring atoms N5 and C4A are supposed to be the most chemically active with respect to different substrates. In the oxidized form, N5 and C4A atoms are the most common targets for nucleophilic attack. In the reduced states, atoms N5 and C4A are likely to be subject to electrophilic attack. Covalent adducts to C4A of flavin are frequently found as reaction intermediates. Another essential determinant for the reactivity is the mutual orientation of the substrate and the isoalloxazine ring in the Michaelis complex. Hydrogen bonds between protein atoms and flavin N1, N3, O2, and O4 atoms could significantly influence the reactivity of the isoalloxazine ring.

3.6.Interaction between flavins and the protein

There are more than 730 entries for FAD- and 350 entries for FMN-containing proteins in the Protein Data Bank (PDB) {Berman, 2000 #186}). A systematic analysis of the interaction between the flavin isoalloxazine ring and the apo-protein moiety reveal that the isoalloxazine ring of FMN tends to interact with protein side-chain atoms, whilst the isoalloxazine ring of FAD interacts primarily with protein main-chain atoms {Senda, 2009 #2471}. In the case of FAD, atoms O2, N3, and O4 interact mostly with main-chain protein atoms. However, side-chain and main-chain atoms contribute equally to interactions with O2, N3, and O4 of FMN. Because of the fact that main-chain protein atoms normally are less mobile than side-chain ones, the results suggest that the FAD isoalloxazine ring can be more firmly fixed to the protein than that one of FMN. N1 and N5 atoms of the FAD isoalloxazine ring show a larger tendency to interact with side-chain atoms than atoms O2, N3, and O4. The protonation states of the above two atoms (koi) are very sensitive to the redox states of the flavin and the pH of the solution. An alteration of the protonation states of N1 and N5 atoms could cause a reorganization of the hydrogen bonding system around the isoalloxazine ring. Taking into account that protein's side chain is generally more flexible than the main chain, the interactions of N1 and N5 atoms with side-chain atoms could be suitable for the reorganization of the hydrogen bond network. This reorganization could additionally promote conformational changes in the entire protein. The solvent-accessible surface (SAS) is commonly used as a quantitative marker to evaluate the environment of specific atoms in a macromolecule. Almost 95% of the isoalloxazine rings of FAD in the flavoproteins available in the PDB are mostly hidden in the protein matrix with <10% of the solvent SAS. This finding indicates that changes in conformation and ionisation states of the isoalloxazine ring of FAD could drive changes in the protein structure. The isoalloxazine ring of FMN showed the partial accessible surface area of <10% for 75% of the FMN-containing proteins {Senda, 2009 #2471}. In contrast, the isoalloxazine moiety of the FMN of a most of proteins is accessible from the protein surroundings. The interactions between the phosphate moiety of flavin and the protein also have a regular profile.

Analysis of crystal structures of FMN-containing proteins in the PDB showed that often the phosphate moiety of FMN is narrowly spaced to the N-terminal end of the α-helix. The negative charge of the phosphate may well stabilize the interaction with the α-helix {Lostao, 2000 #2472}. The phosphate oxygens could also interact with amide protons as well positively charged side chains.

The interactions between the pyrophosphate moiety and the protein-based positive charges were also suggested to contribute significantly in stabilizing FAD binding. Four types of FAD-binding folds {Dym, 2001 #2473}, were proposed and each of them has a conserved motif to interact with the pyrophosphate. The negative charges of the pyrophosphate can interact favorably with positively charged amino acids, main chain peptide and total a-helices. The adenosine moiety of FAD might also stabilize the cofactor binding.

3.7.Conformation of the isoalloxazine ring in the protein

An important determinant for the isoalloxazine's ring conformation can be its bending angle. An analysis of the isoalloxazine bending angle showed that for all flavoproteins in PDB it varies from 0° to 34°{Senda, 2009 #2471}. The biggest bending angle was found for thioredoxin reductase. For the majority of flavoproteins this angle is less than 10°, or the structure is almost nearly planar. There still are large amount of flavoproteins bending angles >10°. It is worth mentioning that some flavoeznymes contain oxidized flavin with a bent isoalloxazine moiety. For example monoamine oxidases {Ma, 2004 #2474}{Binda, 2002 #2475}, polyamine oxidase {Binda, 1999 #2476} and cholesterol oxidase {Li, 2002 #2477}.

NMR analyses of isoalloxazine-ring conformations and their interactions with proteins was performed and a method to detect the conformations of flavin and its interactions with proteins using 13C and 15N NMR spectroscopy was proposed {Pust, 2002 #2478},{SANNER, 1991 #2479}.

The analysis suggests that the hybridization of N10 and N5 atoms of the isozlloxazine ring in many flavin proteins such us riboflavin-binding protein {Moonen, 2002 #2480}, old yellow enzyme, p-hydroxybenzoate hydroxylase {VERVOORT, 1991 #2481}, electron-transfer flavoprotein{Griffin, 1998 #2482}, flavocytochrome b2{Fleischmann, 2000 #2483}, and thioredoxin reductase {Lennon, 1999 #2484} alters from sp2 type to sp3 type under reduction. The bending angle of the isoalloxazine rings of old yellow enzyme, p-hydroxybenzoate hydroxylase, flavocytochrome b2, and thioredoxin reductase in solution conditions were confirmed by their X-ray structures.

According to X-ray crystallography the redox-dependent conformational changes of the isoalloxazine ring of flavins were also proved. For example, thioredoxin reductase {Lennon, 1999 #2484}, mercury reductase {Ledwidge, 2005 #2485}, proline utilization A (PutA) {Zhang, 2006 #2486}, and ferredoxin reductase BphA4 {Senda, 2007 #2487} exhibit such redox-dependent conformational changes. For several flavoproteins such conformational changes of the isoalloxazine ring are supposed to stimulate additional conformational changes in the protein.