Flavin enzymes Essay

Chapter 3 Flavin Enzymes: Biological Functions, Structure and Interactions

3.1. Introduction

Flavin-containing enzymes are category of enzymes which contain the flavin isoalloxazine heterocyclic ring as a cofactor. This cofactor can either be flavin mononucleotide ( FMN ) or flavin A dinucleotie ( FAD ) . They both are constituted from vitamin B2: riboflavin 5′-phosphate for FMN and an to boot attached adenosine monophosphate for FAD. The nucleotide portion of the flavin ring does non take part in any chemical transmutations, but is of import for acknowledgment and binding of the cofactor to the peculiar enzyme { REFs? } . The chemical expression of the both cofactors is shown in Figure 3.1

Experimental techniques as X-ray crystallography, electronic soaking up spectrometry, round dichroism, and kinetic surveies have been extensively applied in analyzing the flavin-containing enzymes { REFs? } . Experimental consequences provide an first-class background for the application of computational methods which can supply farther important penetrations into atomistic, electronic structural and dynamical belongingss of the flavoenzymes that can non be gained entirely by experiment. Computational chemical science methods have been successfully applied to look into flavin enzymes and in peculiar in uncovering their mechanisms, the function of the flavin cofactor and the consequence of the protein environment { Senn, 2009 # 2559 ; Ridder, 2003 # 1172 ; Mulholland, 2001 # 1114 } .

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A big figure of crystal constructions of FMN and FAD containing flavoproteins are presently available in the protein informations bank ( PDB ) { REF } . It is noteworthy that around 1-3 % of the cistrons in bacterial and eucaryotic genomes encode flavin-binding proteins. The flavoprotein category of enzymes performs a big assortment of biochemical procedures that are involved in of import biological maps such as negatron transportation, dehydrogenation of a diverseness of metabolites, light emanation, activation of O for oxidization { Berg, 2002 # 176 } . Flavoenzymes can besides catalyse unusual chemical reactions, such as halogenation at the 7th place of an aromatic substrate – tryptophan { Murphy, 2006 # 1792 } and oxidative deaminization of biogenic aminoalkanes { Edmondson, 2004 # 1366 } . The combination of structural analyses with experimental 1s such as fast dynamicss and other surveies provides accurate information about the structure-function relationships in flavoenzymes.

The flavin cofactor izoalloxazine ring can be in the undermentioned oxidation-reduction provinces: oxidized and reduced ( one-electron-semiquinone and fully-two-electron-hydroquinone decreased signifier ) ( Figure 3.2 ) { Berg, 2002 # 176 } . The spectroscopic belongingss of the signifiers of the izoalloxazine ring were widely studied and used in disclosure of the reaction mechanisms of flavoenzymes { Senda, 2009 # 2471 } .

3.2.Flavin binding site

The chemically interesting portion of the flavin cofactor is its tricyclic isoalloxazine mediety ( Figure.3.1 ) . Each of the provinces ( oxidation-reduction, ionic, or electronic ) has specific electronic, spectral and chemical belongingss which can be influenced by the protein environment. The flavin isoalloxazine ring is amphipathic and is formed by merger between the hydrophobic dimethylbenzene and the hydrophilic pyrimidine ring. The redox potency of the two-electron decrease of the flavin is about 2200 millivolt. However, this value varies greatly between different flavoenzymes ( e.g. from 2400 millivolt to 160 millivolts 13,14 ) . It is thought that the propinquity of a positive charge increases the redox potency, 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 function in increasing the oxidative potency of the flavin. The isoalloxazine ring may follow conformations that differ from planarity ( e.g. polyamine oxidase, cholesterin oxidase and trimethylamine dehydrogenase { REFs? } ) nevertheless its ability to organize H bonds is exhibited in all enzymes. Flavoenzymes can catalyse reactions through formation of a covalent intermediate formed by the substrate onslaught on the flavin ring. For illustration, such covalent contact action is exhibited by nitroalkane oxidase, which performs the oxidative debasement of nitroalkanes to merchandises such as nitrite, hydrogen peroxide, and a carbonyl compound { Senda, 2009 # 2471 } .

3.3. Redox provinces of flavins

The spectroscopic features of the flavin oxidation-reduction signifiers have been used extensively in uncovering reaction mechanisms of flavoenzymes { new wave Berkel WJ, 1999 # 2470 } . Oxidized signifiers of flavin cofactors show aspecific soaking up near 450 nanometers. The oxidised isoalloxazine ring has two pKa values: ~0 for N1 and ~10 for N3, hence under physiological conditions the oxidised isoalloxazine ring of FAD and FMN is normally in a impersonal signifier. One negatron decrease transforms the oxidised flavin to the semiquinone signifier. The semiquinone has a pKa value of ~8.3 for the N5 atom. The impersonal protonated semiquinone is bluish ( & A ; lambda ; max ~560 nanometer ) and the anionic semiquinone is ruddy ( & A ; lambda ; max 390-410 nanometer and ~480 nanometer ) . Both semiquinone signifiers are reported for assorted flavoenzymes. When the isoalloxazine ring is to the full reduced to a hydroquinone, flavin becomes about colourless ( it is still pale yellow ) . As the hydroquinone has a pKa value of ~6.6 for the N1 atom, the to the full reduced flavin could be in impersonal or anionic signifiers under physiological conditions.

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

The isoalloxazine ring of free oxidised flavins in crystal constructions has a two-dimensional conformation { Fritchie, 1975 # 2560 } , whereas the to the full reduced flavin adopts a dead set conformation along the N5-N10 axis { Werner, 1970 # 2561 } . The conformation of the isoalloxazine ring in crystal constructions can be influenced by steric effects due to crystal wadding. In order to supply penetrations into flavin conformations in solution computational surveies have been performed. Electronic construction computations confirmed that the oxidised isoalloxazine ring is two-dimensional ; nevertheless, it bends along the N5- N10 axis after two-electron decrease. The hybridisation of the N5 and N10 atoms in the to the full reduced flavin were calculated to be intermediate between sp2 and sp3 type. Calculations of the semiquinone signifier of the isoalloxazine pealing predict a conformation really near to planar { Zheng, 1996 # 2565 } . However, NMR experiments of free flavins in solution demonstrate that to the full reduced flavin in solution contains an sp2-hybridized N10 atom, and that the N5 atom besides exhibits a chiefly sp2 character, bespeaking that the to the full reduced flavin is planar in H2O { Moonen, 2002 # 2566 } . The energy barrier for the bent-planar passage of the decreased flavin was estimated by NMR as being & A ; lt ; 4.8 kcal/mol. A theoretical computation besides showed that the energy barrier for the bent-planar passage was merely ~6.4 kcal/mol { Zheng, 1996 # 2565 } . Therefore the isoalloxazine pealing conformation may be sensitive to environmental effects ( such us interactions with the protein environment ) . QM/MM computations which take the effects of the protein environment into history could be really utile in the probe of flavin conformations.

3.5. Responsiveness of flavins in the protein

The oxidation-reduction belongingss of the isoalloxazine pealing supply the ability for FAD and FMN to move as redox-active cofactors in flavoproteins that catalyze a diverse scope of reactions. Isoalloxazine pealing atoms N5 and C4A are supposed to be the most chemically active with regard to different substrates. In the oxidised signifier, N5 and C4A atoms are the most common marks for nucleophilic onslaught. In the decreased provinces, atoms N5 and C4A are likely to be capable to electrophilic onslaught. Covalent adducts to C4A of flavin are often found as reaction intermediates. Another indispensable determiner for the responsiveness is the common orientation of the substrate and the isoalloxazine ring in the Michaelis composite. Hydrogen bonds between protein atoms and flavin N1, N3, O2, and O4 atoms could significantly act upon the responsiveness 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 mediety reveal that the isoalloxazine ring of FMN tends to interact with protein side-chain atoms, whilst the isoalloxazine ring of FAD interacts chiefly with protein main-chain atoms { Senda, 2009 # 2471 } . In the instance of FAD, atoms O2, N3, and O4 interact largely with main-chain protein atoms. However, side-chain and main-chain atoms contribute every bit to interactions with O2, N3, and O4 of FMN. Because of the fact that main-chain protein atoms usually are less nomadic than side-chain 1s, the consequences suggest that the FAD isoalloxazine ring can be more steadfastly fixed to the protein than that one of FMN. N1 and N5 atoms of the FAD isoalloxazine pealing demo a larger inclination to interact with side-chain atoms than atoms O2, N3, and O4. The protonation provinces of the above two atoms ( koi ) are really sensitive to the redox provinces of the flavin and the pH of the solution. An change of the protonation provinces of N1 and N5 atoms could do a reorganisation of the H adhering system around the isoalloxazine ring. Taking into history that protein ‘s side concatenation is by and large more flexible than the chief concatenation, the interactions of N1 and N5 atoms with side-chain atoms could be suited for the reorganisation of the H bond web. This reorganisation could to boot advance conformational alterations in the full protein. The solvent-accessible surface ( SAS ) is normally used as a quantitative marker to measure the environment of specific atoms in a supermolecule. Almost 95 % of the isoalloxazine rings of FAD in the flavoproteins available in the PDB are largely hidden in the protein matrix with & A ; lt ; 10 % of the solvent SAS. This determination indicates that alterations in conformation and ionization provinces of the isoalloxazine ring of FAD could drive alterations in the protein construction. The isoalloxazine ring of FMN showed the partial accessible surface country of & A ; lt ; 10 % for 75 % of the FMN-containing proteins { Senda, 2009 # 2471 } . In contrast, the isoalloxazine mediety of the FMN of a most of proteins is accessible from the protein milieus. The interactions between the phosphate mediety of flavin and the protein besides have a regular profile.

Analysis of crystal constructions of FMN-containing proteins in the PDB showed that frequently the phosphate mediety of FMN is narrowly spaced to the N-terminal terminal of the & A ; alpha ; -helix. The negative charge of the phosphate may good stabilise the interaction with the & A ; alpha ; -helix { Lostao, 2000 # 2472 } . The phosphate Os could besides interact with amide protons as good positively charged side ironss.

The interactions between the pyrophosphate mediety and the protein-based positive charges were besides suggested to lend significantly in stabilising FAD binding. Four types of FAD-binding creases { Dym, 2001 # 2473 } , were proposed and each of them has a conserved motive to interact with the pyrophosphate. The negative charges of the pyrophosphate can interact favourably with positively charged amino acids, chief concatenation peptide and entire a-helices. The adenosine mediety of FAD might besides stabilise the cofactor binding.

3.7.Conformation of the isoalloxazine ring in the protein

An of import determiner for the isoalloxazine ‘s ring conformation can be its flexing angle. An analysis of the isoalloxazine bending angle showed that for all flavoproteins in PDB it varies from 0 & A ; deg ; to 34 & A ; deg ; { Senda, 2009 # 2471 } . The biggest bending angle was found for thioredoxin reductase. For the bulk of flavoproteins this angle is less than 10 & A ; deg ; , or the construction is about about two-dimensional. There still are big sum of flavoproteins flexing angles & A ; gt ; 10 & A ; deg ; . It is deserving adverting that some flavoeznymes contain oxidised flavin with a dead set isoalloxazine mediety. For illustration 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 observe the conformations of flavin and its interactions with proteins utilizing 13C and 15N NMR spectrometry was proposed { Pust, 2002 # 2478 } , { SANNER, 1991 # 2479 } .

The analysis suggests that the hybridisation of N10 and N5 atoms of the isozlloxazine ring in many flavin proteins such us riboflavin-binding protein { Moonen, 2002 # 2480 } , old xanthous 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 decrease. The flexing angle of the isoalloxazine rings of old xanthous enzyme, p-hydroxybenzoate hydroxylase, flavocytochrome b2, and thioredoxin reductase in solution conditions were confirmed by their X-ray constructions.

Harmonizing to X-ray crystallography the redox-dependent conformational alterations of the isoalloxazine ring of flavins were besides proved. For illustration, thioredoxin reductase { Lennon, 1999 # 2484 } , mercury reductase { Ledwidge, 2005 # 2485 } , proline use A ( PutA ) { Zhang, 2006 # 2486 } , and ferredoxin reductase BphA4 { Senda, 2007 # 2487 } exhibit such redox-dependent conformational alterations. For several flavoproteins such conformational alterations of the isoalloxazine ring are supposed to excite extra conformational alterations in the protein.


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