The Flavoprotein Enzymes

The flavoproteins, or yellow enzymes, have been shown to contain either alloxazine mononucleotide (flavin mononucleotide, FMN) or isoalloxazine adenine dinucleotide (flavin adenine dinucleotide, FAD) (Fig. 6). The 

Flavoproteins occur as oxidases which catalyze the direct oxidation of various substrates by oxygen. They appear to be of major importance, 

Theorell obtained the enzyme in essentially pure form by electrophoretic separation. The prosthetic group was FMN, and the absorption spectrum showed bands at 380 and 465 m/x which disappeared on reduction. From the flavin content the molecular weight was estimated to be about 80,000, a value which was confirmed by ultracentrifugal measurements.  

Theorell succeeded in dissociating the prosthetic group of old yellow enzyme from the protein by means of dialysis against dilute acid. The active enzyme was regenerated when the two components were mixed. Later Warburg and Christian introduced a more generally applicable method for this purpose which involved acidification to pH 1 to 3 in the presence of ammonium sulfate. The protein component is precipitated, and the flavin remains in solution. This procedure for the reversible separation of protein and prosthetic group has been successfully applied to many of the known flavoprotein enzymes. 

The role of the old yellow enzyme in metabolism is not known. It is reduced by TPNH2 or DPNH2 and the reduced form is reoxidized by atmospheric oxygen. Its activity in this system, however, is too low to account for more than a fraction of yeast respiration. It is more rapidly reoxidized by methylene blue, but very slowly, if at all, by cytochrome c. 

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The pKa assignments, which have been controversial, are discussed in Chapter 7. This disulfide loop is reduced by NADPH through the action of the flavoprotein enzyme thioredoxin reductase. 

Those include C-, N- and 5-oxidations, N-, 0- and 5-deaIkylation, deaminations, and certain dehalogenations. Under anaerobic conditions, it can also catalyze reductive reactions. The CYP monooxygenase system is a multien-zymatic complex constituted by the CYP hemoprotein, the flavoprotein enzyme NADPH CYP reductase, and the unsatnrated phospholipid phosphatidylcholine. The isoforms involved in xenobiotic metabolism are membrane bonnd enzymes situated in the endoplasmic reticnlnm. After... 

In the first part of this book devoted to cellular metabolism, it was pointed out that riboflavin is converted to monophosphate and diphosphate, yielding the active coenzymes of a large number of enzymes involved in the cellular oxidation-reduction reaction. As can be expected, the enzyme activity for which these flavoproteins are responsible is decreased in riboflavin deficiency. This has been clearly demonstrated for amino acid oxidase. Extensive studies on the effect of riboflavin deficiency on a large number of proteins have not been carried out but it seems logical to assume that the clinicopathological changes that develop in riboflavin deficiency result from the reduced activity of the flavoprotein enzyme. Yet, many... 

The first step in the )8-oxidation cycle proper is the introduction of a tram-a,p double bond into the hydrocarbon chain of the activated fatty acid, catalysed by the flavoprotein enzyme, acyl-CoA dehydrogenase. 

In contrast to the nicotinamide nucleotide dehydrogenases, the prosthetic groups FMN and FAD are firmly associated with the proteins, and the flavin groups are usually only separated from the apoen2yme (protein) by acid treatment in water. However, in several covalently bound flavoproteins, the enzyme and flavin coen2ymes are covalently affixed. In these cases, the flavin groups are isolated after the proteolytic digestion of the flavoproteins. 

Flavoprotein enzymes contain flavin mononucleotide (FMN) or flavin adenine dinucleotide (FAD) as prosthetic groups. FMN and FAD are formed in the body from the vitamin riboflavin (Chapter 45). FMN and FAD are usually tighdy—but not covalendy—bound to their respecdve apoenzyme proteins. Metalloflavopro-teins contain one or more metals as essential cofactors. 

Examples of flavoprotein enzymes include L-amino acid oxidase, an FMN-finked enzyme found in kidney with general specificity for the oxidadve deamination of... 

A modified form of P-oxidation is found in peroxisomes and leads to the formation of acetyl-CoA and H2O2 (from the flavoprotein-linked dehydrogenase step), which is broken down by catalase. Thus, this dehydrogenation in peroxisomes is not linked directly to phosphorylation and the generation of ATP. The system facilitates the oxidation of very long chain fatty acids (eg, Cjq, C22). These enzymes are induced by... 

Although reduction of chromate Cr to Cr has been observed in a number of bacteria, these are not necessarily associated with chromate resistance. For example, reduction of chromate has been observed with cytochrome Cj in Desulfovibrio vulgaris (Lovley and Phillips 1994), soluble chromate reductase has been purified from Pseudomonas putida (Park et al. 2000), and a membrane-bound reductase has been purified from Enterobacter cloacae (Wang et al. 1990). The flavoprotein reductases from Pseudomonas putida (ChrR) and Escherichia coli (YieF) have been purified and can reduce Cr(VI) to Cr(III) (Ackerley et al. 2004). Whereas ChrR generated a semi-quinone and reactive oxygen species, YieR yielded no semiquinone, and is apparently an obligate four-electron reductant. It could therefore present a suitable enzyme for bioremediation. 

Activity improvement via co-expression or supplementation of the fourth enzyme DszD, the flavoprotein which provided reduced co-factor to DszC and DszA, was found to be successful [297,298], [126,299], Additionally, providing nutrient broth to favor the microorganism growth was also successful in activity improvement [300],... 

The reaction-center proteins for Photosystems I and II are labeled I and II, respectively. Key Z, the watersplitting enzyme which contains Mn P680 and Qu the primary donor and acceptor species in the reaction-center protein of Photosystem II Qi and Qt, probably plastoquinone molecules PQ, 6-8 plastoquinone molecules that mediate electron and proton transfer across the membrane from outside to inside Fe-S (an iron-sulfur protein), cytochrome f, and PC (plastocyanin), electron carrier proteins between Photosystems II and I P700 and Au the primary donor and acceptor species of the Photosystem I reaction-center protein At, Fe-S a and FeSB, membrane-bound secondary acceptors which are probably Fe-S centers Fd, soluble ferredoxin Fe-S protein and fp, is the flavoprotein that functions as the enzyme that carries out the reduction of NADP+ to NADPH. 

A flavoprotein that accepts reducing equivalents and transfers them to the catalytic enzyme... 

MAO is a flavoprotein enzyme that is found on the outer membrane of mitochondria. It oxidatively deami nates short-chain monoamines only, and it is not part of the DM MS. ATP is involved in the transfer of reducing equivalents through the mitochondrial respiratory chain, not the microsomal system. 

Molybdenum (Mo) is present in all plant, human, and animal tissues, and is considered an essential micronutrient for most life forms (Schroeder et al. 1970 Underwood 1971 Chappell and Peterson 1976 Chappell et al. 1979 Goyer 1986). The first indication of an essential role for molybdenum in animal nutrition came in 1953 when it was discovered that a flavoprotein enzyme, xanthine oxidase, was dependent on molybdenum for its activity (Underwood 1971). It was later determined that molybdenum is essential in the diet of lambs, chicks, and turkey poults (Underwood 1971). Molybdenum compounds are now routinely added to soils, plants, and waters to achieve various enrichment or balance effects (Friberg et al. 1975 Friberg and Lener 1986). 

Nicotinic acid derivatives occur in biologic materials as the free acid, as nicotinamide, and in two coenzymatic forms nicotinamide adenine dinucleotide (NAD), and nicotinamide adenine dinucleotide phosphate (NADP). These coenzymes act in series with flavoprotein enzymes and, like them, are hydrogen acceptors or, when reduced, donors. Several plants and bacteria use a metabolic pathway for the formation of nicotinic acid that is different from the tryptophan pathway used by animals and man (B39). 

Figure 9.1 CYP catalytic cycle. The sequential two-electron reduction of CYP and the various transient intermediates were first described in the late 1960s [206], The sequence of events that make up the CYP catalytic cycle is shown. The simplified CYP cycle begins with heme iron in the ferric state. In step (i), the substrate (R—H) binds to the enzyme, somewhere nearthe distal region of the heme group and disrupts the water lattice within the enzymes active site [207], The loss of water elicits a change in the heme iron spin state (from low spin to high spin) [208]. Step (ii) involves the transfers of an electron from NADPH via the accessory flavoprotein NADPH-CYP reductase, with the electron flow going from the reductase prosthetic group FAD to FMN to the CYP enzyme [206,209]. The...

Lynen had studied chemistry in Munich under Wieland his skill as a chemist led to the successful synthesis of a number of fatty acyl CoA derivatives which proved to be substrates in the catabolic pathway. Many of these C=0 or C=C compounds had characteristic UV absorption spectra so that enzyme reactions utilizing them could be followed spectrophotometrically. This technique was also used to identify and monitor the flavoprotein and pyridine nucleotide-dependent steps. Independent evidence for the pathway was provided by Barker, Stadtman and their colleagues using Clostridium kluyveri. Once the outline of the degradation had been proposed the individual steps of the reactions were analyzed very rapidly by Lynen, Green, and Ochoa s groups using in the main acetone-dried powders from mitochondria, which, when extracted with dilute salt solutions, contained all the enzymes of the fatty acid oxidation system. 

The cases of myoglobin and hemoglobin are not rare. Many enzymes are dependent for their function on the presence of a nonprotein group. For example, cytochrome c also contains a prosthetic group similar, but not identical, to heme, as do a number of other proteins. These are known generically as heme proteins. There is a family of enzymes that contain a flavin group, the flavoproteins. Another family contains pyridoxal phosphate, a derivative of vitamin Be. There are a number of other examples. 

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