Flavin coenzymes reduced

Flavin coenzymes can exist in any of three different redox states. Fully oxidized flavin is converted to a semiqulnone by a one-electron transfer, as shown in Figure 18.22. At physiological pH, the semiqulnone is a neutral radical, blue in color, with a A ax of 570 nm. The semiqulnone possesses a pAl of about 8.4. When it loses a proton at higher pH values, it becomes a radical anion, displaying a red color with a A ax of 490 nm. The semiqulnone radical is particularly stable, owing to extensive delocalization of the unpaired electron across the 77-electron system of the isoalloxazine. A second one-electron transfer converts the semiqulnone to the completely reduced dihydroflavin as shown in Figure 18.22. 

Direct hydroxylation of an aromatic ring to yield a hydroxybenzene (a phenol) is difficult and rarely done in the laboratory., but occurs much more frequently in biological pathways. An example is the hydroxylation of p-hydroxyphenyl acetate to give 3,4-dihydroxyphenyl acetate. The reaction is catalyzed by p-hydroxyphenylacctate-3-hydroxylase and requires molecular oxygen plus the coenzyme reduced flavin adenine dinucleotide, abbreviated FADH2. 

B. Nicotinamide and Flavin Coenzymes.—High-frequency (220 MHz) H n.m.r. spectroscopy shows that there are differences in conformation between oxidized and reduced pyridine coenzymes. A preliminary report on the P n.m.r. spectra of NAD+ and NADH confirms these observations, as the spectrum of NAD+ consists of an AB quartet while there is only a single resonance discernible in the spectrum of NADH. 

Although the reduction potentials of DNA bases and UV induced DNA lesions inside a DNA double strand or inside the active site of a DNA photolyase, together with the reduction potential of the photoexcited FADH- in the photolyases, are not known, currently available redox potentials indicate that the single electron reduction of a nucleobase or a UV induced dimer lesion by a reduced and deprotonated flavin coenzyme is a weakly exothermic process. The reduced and deprotonated FADH- in its photoexcited state is... 

Scheme 2 Mechanism of repair of cyclobutane pyrimidine dimers (CPD) by a CPD photolyase. 8-HDF 8-hydroxy-5-deazaflavin, ET electron transfer. FADH reduced and de-protonated flavin-coenzyme...

Indicine IV-oxide (169) (Scheme 36) is a clinically important pyrrolizidine alkaloid being used in the treatment of neoplasms. The compound is an attractive drug candidate because it does not have the acute toxicity observed in other pyrrolizidine alkaloids. Indicine IV-oxide apparently demonstrates increased biological activity and toxicity after reduction to the tertiary amine. Duffel and Gillespie (90) demonstrated that horseradish peroxidase catalyzes the reduction of indicine IV-oxide to indicine in an anaerobic reaction requiring a reduced pyridine nucleotide (either NADH or NADPH) and a flavin coenzyme (FMN or FAD). Rat liver microsomes and the 100,000 x g supernatant fraction also catalyze the reduction of the IV-oxide, and cofactor requirements and inhibition characteristics with these enzyme systems are similar to those exhibited by horseradish peroxidase. Sodium azide inhibited the TV-oxide reduction reaction, while aminotriazole did not. With rat liver microsomes, IV-octylamine decreased... 

Figure 5.3 The flavin coenzymes FAD and FMN. Note that in contrast to NAD+, flavins can be half-reduced to the stable radical FADH or fully reduced to the dihydroflavin shown.

Flavin Coenzymes.—5-Deazaflavin-adenine dinucleotide (2) can be prepared from the 5-deazaFMN,21 using a FAD pyrophosphorylase from rat liver.22 When the apoprotein of D-amino-acid oxidase from pig kidney is reconstituted with (2), no oxidation of D-alanine is observed, although the flavin chromophore in the reconstituted enzyme is reduced on the addition of DL-amino-acids.22 This has been interpreted as indicating that hydrogen transfer from the amino-acid to (2) can still... 

Autofluorescence of cells often complicates the studies with fluorescence microscopy (especially the application of green fluorescent substances). There are different reasons for the occurrence of this phenomenon (157) (i) the fluorescent pigment lipofuscin, which settles with rising age in the cytoplasm of cells (ii) cell culture medium, which often contains phenol red that increases autofluorescence (iii) endogen substances such as flavin coenzymes [flavin-adenine dinucleotide (FDA), flavin mononucleotide (FMN) absorp-tion/emission 450/515nm], pyridine nucleotides [reduced nicotinamide adenine dinucleotide (NADH) absorption/emission 340/460nm] or porphyrine (iv) substances taken up by cells (as mentioned above filipin) and (v) preparation of the cells fixation with glutaraldehyde increases autofluorescence. 

All the internal monooxygenases that have so far been purified and characterized contain flavin coenzymes. The external hydrogen donors include reduced NAD, reduced NADP, ascorbic acid and sulfhydryl compounds. Cofactors required for the external monooxygenases are flavin, pteridine, copper, nonheme iron and heme as cytochrome P-450. In some monooxygenase reactions, enzymes and/or electron carrier systems other than monooxygenase itself are involved in the transfer of an electron or hydrogen from the external hydrogen donor to the cofactor involved. 

Why are there four major hydrogen transfer coenzymes, NAD+, NADP+, FAD, and riboflavin phosphate (FMN), instead of just one Part of the answer is that the reduced pyridine nucleotides NADPH and NADH are more powerful reducing agents than are reduced flavins (Table 6-7). Conversely, flavin coenzymes are more powerful oxidizing agents than are... 

The attention of biochemists was first attracted to flavins as a result of their color and fluorescence. The study of spectral properties of flavins (Fig. 15-8) has been of importance in understanding these coenzymes. The biochemical role of the flavin coenzymes was first recognized through studies of the "old yellow enzyme"144 145 which was shown by Theorell to contain riboflavin 5 -phosphate. By 1938, FAD was recognized as the coenzyme of a different yellow protein, D-amino acid oxidase of kidney tissue. Like the pyridine nucleotides, the new flavin coenzymes were reduced by dithionite to nearly colorless dihydro forms (Figs. 15-7 and 15-8) revealing the chemical basis for their function as hydrogen carriers. 

Flavin coenzymes are usually bound tightly to proteins and cycle between reduced and oxidized states while attached to the same protein molecule. In a free unbound coenzyme the redox potential is determined by the structures of the oxidized and reduced forms of the couple. Both riboflavin and the pyridine nucleotides contain aromatic ring systems that are stabilized by resonance. Part of this resonance stabilization is lost upon reduction. The value of E° depends in part upon the varying amounts of resonance in the oxidized and reduced forms. The structures of the coenzymes have apparently evolved to provide values of E° appropriate for their biological functions. 

PQQ and the other quinone prosthetic groups described here all function in reactions that would be possible for pyridine nucleotide or flavin coenzymes. All of them, like the flavins, can exist in oxidized, half-reduced semiquinone and fully reduced dihydro forms. The questions to be asked are the same as we asked for flavins. How do the substrates react How is the reduced cofactor reoxidized In nonenzymatic reactions alcohols, amines, and enolate anions all add at C-5 of PQQ to give adducts such as that shown for methanol in Eq. 15-51, step a 444,449,449a Although many additional reactions are possible, this addition is a reasonable first step in the mechanism shown in Eq. 15-51. An enzymatic base could remove a proton as is indicated in step b to give PQQH2. The pathway for reoxidation (step c) might involve a cytochrome b, cytochrome c, or bound ubiquinone.445 446... 

Flavin adenine dinucleotide. See FAD Flavin adenine diphosphate. See FAD Flavin coenzymes 766,780 - 795 modified 788, 789 reduced 794 Flavin radicals 792 color of 794 formation constant 794 Flavocytochrome b2 782, 794, 847 Flavodoxins 793, 799, 800 Flavoprotein(s) 513, 788... 

Fig. 6. Oxidized and reduced states of flavin coenzymes. R represents the remainder of the coenzyme as given in Fig. 4...

Flavin coenzymes exist in three spectrally distinguishable oxidation states that account in part for their catalytic functions the yellow oxidized form, the red or blue one-electron reduced form, and the colorless two electron re-... 

The functional end of the flavin coenzymes FMN and FAD is the tricyclic isoalloxazine system, with the numbering system shown in structure I, the air-stable, yellow, oxidized form. The other two functionally important redox states are the one-electron-reduced semiquinone, II (pKa = 8.4 for dissociation at N(5)), and the two-electron-reduced, colorless dihydroflavin, III. In the dihydro form N(5), C(4a), C(la), andN(l) form a diaminoethylene system and it was anticipated that nitrogen at the 5 and 1 positions would be key to coenzymatic function. 

Biological autofluorescence in mammalian cells due to flavin coenzymes (FAD and FMN absorption, 450 nm emission, 515 nm) and reduced pyridine nucleotides (NADH absorption, 340 nm emission, 460 nm) can be problematic in the detection of fluorescence probes in tissues and cells. Fixation with aldehydes, particularly glutaraldehyde, can result in high levels of autofluorescence. This can be minimized in fixed cells by washing with 0.1% sodium borohydride in phosphate-buffered saline (5) prior to antibody incubation. Problems due to autofluorescence can be minimized by selecting probes and optical filters that maximize the fluorescence signal relative to the autofluorescence. Other factors that limit IF include the performance of the detection instrument (i.e. how well the microscope has been calibrated and set), the specificity of the antibodies, and the specimen preparation. 

In bacterial NADHrFMN oxidoreductase [15], the flavin coenzyme is bound relatively weakly. This enzyme serves to provide reduced FMN to luciferase in... 

Reoxidation of reduced flavin coenzymes is the major source of oxygen radicals in the body, and riboflavin is also capable of generating reactive oxygen species nonenzymically. As protection against this, there is very strict control over the body content of riboflavin. Absorption is limited, and any in excess of requirements is rapidly excreted. 

The flavin coenzyme occurs in each of the oxidases discussed here as flavin adenine dinucleotide (FAD). The R group attached to is adenosyldiphosphoribityl. The flavin nucleus can exist in three redox states, each of which can adopt three ionization states (8, 9). Only two redox states—fully oxidized and fully reduced see Equation 1)—are kinetically important in the simple flavoprotein oxidases under discussion. 

A final distinction from nicotinamides is that the flavin coenzymes generally form tight non-dissociable non-covalent complexes with the apoenzyme. Nicotinamides are released at the end of each catalytic cycle and so are consumed as substrate as part of the redox stoichiometry. Because flavins are tightly bound to the apoprotein (/irD= 10 -10 " M) the coenzyme must be oxidised/reduced at the end each turnover before the enzyme complex again becomes catalytically active. Differential binding of flavin and dihydroflavin is responsible for the wide range of redox potentials for flavoproteins so that oxidation or reduction can be thermodynamically favourable. For example, D-amino acid oxidase binds FAD with a dissociation constant of 10 M but FADHj with one of 10 M which changes the reduction potential from —200 for the FAD/FADHj couple free in solution to 0 mV when bound to the enzyme. 

In nature, lysine-bound lipoic acid is used to oxidize thiamine-bound acetyldehyde to acetic acid. The reduced dithiol is then reoxidized to the disulfide by a flavin. The lysine oligomethylene chain is thought to act as a pendulum string, when lipoic swings back and forth betwen thiamine and flavin coenzymes... 

FIGURE 15.4 The structures of riboflavin, flavin mononucleotide (FMN), and flavin adenine dinucleotide (FAD). Even in organisms that rely on the nicotinamide coenzymes (NADH and NADPH) for many of their oxidation-reduction cycles, the flavin coenzymes fill essential roles. Flavins are stronger oxidizing agents than NAD and NADP. They can be reduced by both one-electron and two-electron pathways and can be reoxidized easily by molecular oxygen. Enzymes that use flavins to carry out their reactions—flavoenzymes—are involved in many kinds of oxidation-reduction reactions. 

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