Single-Electron-Transferring Flavoproteins

Flavoproteins catalyzing single -electron transfer provide the link between substrate oxidation catalyzed by dehydrogenases and the mitochondrial electron transport chain. 

The reduction of cytochrome P450 hy NADPH involves a single enzyme, NADPH-cytochrome P450 reductase, which contains both FAD and riboflavin phosphate. The FAD undergoes a two-electron reduction at the expense of NADPH, then transfers electrons singly to the riboflavin phosphate, which in turn reduces cytochrome P450. The semiquinone radicals of both FAD and riboflavin phosphate are intermediates in this reaction. 

A distinct electron transfer flavoprotein (ETF) is the single-electron acceptor for a variety of flavoprotein dehydrogenases, including acyl CoA, glutaryl CoA, sarcosine, and dimethylglycine dehydrogenases. It then transfers the electrons to ETF-ubiquinone reductase, the iron-sulfur flavoprotein that reduces ubiquinone in the mitochondrial electron transport chain. 

The initial step of the two-electron-tremsferring reactions is the removal of a proton from the substrate, followed by the intermediate formation of em adduct between the substrate and prosthetic group at N-5 of the flavin. This undergoes cleavage to yield dihydroflavin emd the oxidized product, which is commonly a carbon-Ccubon double bond. The reduced flavin is then reoxidized by reaction with an electron-tremsferring flavoprotein, as discussed above, or in some cases by reaction with nicotineimide nucleotide coenzymes. 

The nicotinamide nucleotide independent flavoprotein dehydrogeneises include the following  

Succinate dehydrogenase in the trictu boxylic acid cycle, which reacts directly with ubiquinone in the mitochondrial electron transport chain. 

Acyl CoA dehydrogenases in fatty acid /3-oxidation. These enzymes are especially sensitive to riboflavin depletion, and riboflavin deficiency is characterized by impaired fatty acid oxidation and organic aciduria (Section 7.4.1). These are also the enzymes affected in riboflavin-responsive organic acidurias. 

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The enzyme is organized into two structural domains,201 one of which binds FAD and the other NADP+. Similar single-electron transfers through flavoproteins also occur in many other enzymes. Chorismate mutase, an important enzyme in biosynthesis of aromatic rings (Chapter 25), contains bound FMN. Its function is unclear but involves formation of a neutral flavin radical.276 277... 

The mechanism by which amines are oxidised by flavoproteins has been an issue of considerable debate in recent years. The debate has been particularly heated in the case of the enzyme monoamine oxidase (Silver-man, 1995). Through the use of a variety of mechanism-based inhibitors and based on studies of nonenzymic mechanisms of amine oxidation, a mechanism for monoamine oxidase in which substrate is initially oxidized by single electron transfer to the enzyme flavin to give an aminium cation... 

The oxidation of diphenols to quinones is reversible, a variety of cellular reductants are able to mediate the reduction of quinones either by a two-electron mechanism or by two single-electron steps. The two-electron reduction can be catalyzed by carbonyl reductase and quinone reductase, while cytochrome P450 and some flavoproteins act by single-electron transfers. The non-enzymatic reduction of quinones can occur, for example, in the presence of O2 or some thiols such as GSH. 

Superoxide is produced by the NADPH oxidoreduc-tase (oxidase), which is a membrane-bound enzyme complex containing a flavoprotein that catalyses the transfer of single electrons from NADPH in the cytosol to extracellular oxygen. NADPH is mainly supplied by the hexose monophosphate shunt. In resting cells, the oxidase complex is inactive and disassembled, but is rapidly reconstituted and activated by chemotactic mechanisms or phagocytosis (Baggiolini and Thelen, 1991). 

In summary, organisms can use biological reductants such as NAD(P)H, capable of hydride transfer (two electron transfer), and reduced flavoproteins and metallo-proteins, capable of single electron donation. Although not necessarily intended to interact with xenobiotic organic compounds, when such organic chemicals come in contact with suitably reactive bioreductants in vivo, reductions can occur. 

The soluble electron carriers released from the reaction centers into the cytoplasm of bacteria or into the stroma of chloroplasts are reduced single-electron carriers. Bacterial ferredoxin with two Fe4S4 clusters is formed by bacteria if enough iron is present. In its absence flavodoxin (Chapter 15), which may carry either one or two electrons, is used. In chloroplasts the carrier is the soluble chloroplast ferredoxin (Fig. 16-16,C), which contains one Fe2S2 center. Reduced ferredoxin transfers electrons to NADP+ (Eq. 15-28) via ferredoxin NADP oxidoreductase, a flavoprotein of known three-dimensional structure.367 369... 

Some of the catalytic and structural properties of thioredoxin reductase as they relate to analogous properties of lipoamide dehydrogenase and glutathione reductase have been covered in Section II. The flavoprotein, thioredoxin reductase, catalyzes the electron transfer from NADPH to thioredoxin, a protein of 12,000 molecular weight containing a single disulfide. The reductase has a reactive disulfide in addition to FAD. Thus, electron flow is from NADPH to the PAD-disulfide system of thioredoxin reductase, to the disulfide of thioredoxin, and finally to a variety of acceptor systems. 

Electron transfer between pyridine nucleotides and disulfide compounds is catalyzed by several flavoproteins and three of these are well characterized. Lipoamide dehydrogenase functions in the oxidative decarboxylation of a-keto acids catalyzing the reoxidation of reduced lipoate by NAD+ 18, 19). Glutathione reductase catalyzes electron transfer between NADPH and glutathione (20-22). Thioredoxin reductase catalyzes the reduction of thioredoxin by NADPH (8) thioredoxin is a protein of 12,000 molecular weight containing a single cystine residue which is the electron acceptor (23). 

The two-domain, structural motif in FNR represents a common structural feature in a large class of enzymes that catalyze electron transfer between a nicotinamide dinucleotide molecule and a one-electron carrier. Beside the photosynthetic electron-transfer enzyme, others non-photosynthetic ones include flavodoxin reductase, sulfite reductase, nitrate reductase, cytochrome reductase, and NADPH-cyto-chrome P450 reductase. FNR belongs to the group of so-called dehydrogenases-electron transferases, i.e., flavoproteins that catalyze electron transfer from two, one-electron donor molecules to a single two-electron acceptor molecule. 

Flavins are unique coenzymes that are able to catalyze both one- and two-electron transfers. Because of this, many flavoproteins are involved in transferring electrons between other proteins. Often, flavoproteins are reduced by two-electron donors, such as pyridine nucleotides, and then pass those electrons one at a time to a single-electron acceptor, such as an iron-sulfur cluster in another protein. Conversely, some enzymes accept single electrons from reduced enzymes. In either case, the flavoenzymes are transferring single electrons thus, flavin semiquinone is frequently stabilized and observed during turnover. 

Flavins are widely recognized by their ability of participate in both one- and two-electron transfer processes, since these compounds can exist in three different redox states oxidized (quinone), one-electron reduced (semiquinone) and two-electron reduced (hydroquinone). The redox potential for the complete reduction of oxidized flavins is about -200 mV, but this value may largely vary in flavoproteins, as a consequence of the protein activity site environment, ranging from —400 mV to +60 mV (Fraaije and Mattevi 2000). Flavins may transfer single electrons, hydrogen atoms and hydride ions. In addition, N5 and C4a of the oxidized flavin molecule are susceptible sites for... 

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