Semiquinone reductases

Fig. 3.12B shows a more detailed molecular interpretation of the 6 cycle. A single ubiquinone species in a ubiquinone pocket of the Complex III monomer is shown to interact either with the quinol oxidase site (o), or with a semiquinone reductase site (i). Flip-flop of the headgroup (usually of SQ ) between the sites might take place, in principle as proposed for bulk phase ubiquinone (see Section 4.7.2). However, here it occurs within a specific proteinaceous pocket so that regulation by the enzyme is possible. 

Chloroplast envelope membranes. The two envelope membranes that surround chloroplasts contain numerous enzyme systems some of which are involved in the formation and reduction of semiquinone radicals. These include quinol oxidase and NADPHiquinone/semiquinone reductases. These reactions are one step in more complex reactions including the biosynthesis of specific plastid membrane constituents and the formation of polyunsaturated fatty adds. Some of these enzymes are also involved in the export of protons to the cytosol, which partially regulates the stromal pH during photosynthesis. 

The addition of sulfite to APS reductase results in changes of the flavin visible spectrum that are explained by the formation of an adduct between the sulfite and the FAD group (135). Addition of AMP to the as-isolated enzyme causes no change in the spectroscopic properties. Addition of AMP to the sulfite-reacted enzyme causes the reduction of center I. However, the formation of a semiquinone signal has never been observed either by EPR or visible spectroscopies. Also, Mossbauer and EPR data indicate that AMP closely interacts with center I (139). 

Dimeric flavoprotein chromate reductases have been purified from Pseudomonas putida (ChrR) and Escherichia coli (YieF). The former produces a semiquinone and transiently reactive oxygen species, whereas the latter is an obligate four-electron reductant. One-electron reduction of Cr(Vl) to Cr(V) has, however, been observed as an intermediate in the reduction by the NAD(P)H-dependent reductase of Pseudomonas ambigua strain G-1 (Suzuki et al. 1992). 

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. 

Component B is a monomeric reductase with a molecular weight of 35,000 and contains per mol of enzyme, 1 mol of FMN, 2.1 mol of Fe, and 1.7 mol of labile sulfur. After reduction with NADH, the ESR spectrum showed signals that were attributed to a [2Fe-2S] structure and a flavo-semiquinone radical (Schweizer et al. 1987). The molecular and kinetic properties of the enzyme are broadly similar to the Class IB reductases of benzoate 1,2-dioxygenase and 4-methoxybenzoate monooxygenase-O-demethylase. 

Qrunones can accept one or two electrons to form the semiquinone anion (Q ") and the hydroquinone dianion (Q ). Single-electron reduction of a quinone is catalyzed by flavoenzymes with relatively low substrate selectivity (Kappus, 1986), for instance NADPH cytochrome P-450 reductase (E.C. 1.6.2.3), NADPH cytochrome b5 reductase (E.C. 1.6.2.2), and NADPH ubiquinone oxidoreductase (E.C. 1.6.5.3). The rate of reduction depends on several interrelated chemical properties of a quinone, including the single-electron reduction potential, as well as the number, position, and chemical characteristics of the substituent(s). The flavoenzyme DT-diphorase (NAD(P)H quinone acceptor oxidoreductase E.C. 1.6.99.2) catalyzes the two-electron reduction of a quinone to a hydroquinone. 

A semiquinone can be readily oxidized to the parent compormd by molecular oxygen and can then re-enter the reductase-catalyzed reaction. The enzymatic reduction and autoxidation of quinones rmder aerobic conditions generates superoxide anion radicals, and this process is known as redox cycling (Figure 2). Flydroquinones are less prone to transfer electrons to oxygen, because the second-electron potential is often too high. 

How the NOS isoforms compare to these related dual flavin enzymes is a matter of ongoing investigation. Characterization of the neuronal NOS revealed that it normally exists in its one-electron reduced form and maintains an air-stable, flavin semiquinone radical (Stuehr and Ikeda-Saito, 1992), as seen for NADPH-cytochrome P450 reductase. It is unknown which flavin in NOS contains the odd electron, although precedent argues that it probably resides on... 

Flavin redox states in a dual flavin enzyme. (Left) Single-electron reduction of the isoalloxazine ring generates the semiquinone radical, while reduction by two electrons generates the fully reduced species. (Right) Five possible oxidation levels of a dual flavin enzyme, where the FMN reduction potential is held at a more positive value relative U) FAD. The flavins can theoretically accept a maximum of four electrons obtained from two NADPH. However, in NADPH-cytochrome P450, reductase, full reduction of the flavins is not normally reached when NADPH serves as the reductant. 

Otvos, J. D., Krum, D. P., and Masters, B. S. (1986). Localization of the free radical on the flavin mononucleotide of the air-stable semiquinone state of NADPH-cytochrome P-450 reductase using 3IP NMR spectroscopy. Biochemistry 25, 7220-7228. 

Nirofuran compounds are also effective anti-parasitic drugs. Nifurtimox, for example, is used to treat Chagas disease (caused by Trypansoma cruzi) but has side effects. In exploring the use of alternatives to nifurtimox, Olea-Azar et al. have examined radical formation from two analogues. Radical anions were observed upon electrolytic reduction of the compounds and a nitroxide, believed to be the glutathionyl radical-adduct, was detected upon electrolysis in the presence of DMPO and GSH. Radical adducts were also detected upon incubation of one of the analogues with microsomes from T. Cruzi.m A novel endo-peroxide reductase has been isolated from T. Cruzi. Whereas the flavoenzyme was found to reduce quinones to their semiquinones, nifurtimox underwent a direct, two-electron reduction, without the formation of radicals.129... 

Pyridine nucleotide-dependent flavoenzyme catalyzed reactions are known for the external monooxygenase and the disulfide oxidoreductases However, no evidence for the direct participation of the flavin semiquinone as an intermediate in catalysis has been found in these systems. In contrast, flavin semiquinones are necessary intermediates in those pyridine nucleotide-dependent enzymes in which electron transfer from the flavin involves an obligate 1-electron acceptor such as a heme or an iron-sulfur center. Examples of such enzymes include NADPH-cytochrome P4S0 reductase, NADH-cytochrome bs reductase, ferredoxin — NADP reductase, adrenodoxin reductase as well as more complex enzymes such as the mitochondrial NADH dehydrogenase and xanthine dehydrogenase. 

Tertiary amine oxides and hydroxy la mines are also reduced by cytochromes P-450. Hydroxylamines, as well as being reduced by cytochromes P-450, are also reduced by a flavoprotein, which is part of a system, which requires NADH and includes NADH cytochrome b5 reductase and cytochrome b5. Quinones, such as the anticancer drug adriamycin (doxorubicin) and menadione, can undergo one-electron reduction catalyzed by NADPH cytochrome P-450 reductase. The semiquinone product may be oxidized back to the quinone with the concomitant production of superoxide anion radical, giving rise to redox cycling and potential cytotoxicity. This underlies the cardiac toxicity of adriamycin (see chap. 6). 

Both the presence of methyl substituents in the tocopherols and their chromanol structures increase the ability of these compounds to form relatively stable radicals.498 499 This ability is doubtless probably important also in the function of ubiquinones and plastoquinones. Ubiquinone radicals (semiquinones) are probably intermediates in mitochondrial electron transport (Chapter 18) and radicals amounting to as much as 40% of the total ubiquinone in the NADH-ubiquinone reductase of heart mito-... 

Berlin, V. and Haseltine, W.A. (1981) Reduction of adriamycin to a semiquinone-free radical by NADPH cytochrome P-450 reductase produces DNA cleavage in a reaction mediated by molecular oxygen, J. Biol. Chem. 256, 4747-4756. 

The ability to form a stable one-electron-reduced radical (semiquinone) allows flavin cofactors to sit at the crossroads of two-electron and one-electron transfer chains. That is, they can be reduced by organic substrates two electrons at a time and be reoxidized by either obligate one-electron acceptors such as cytochromes (e.g., yeast cytochrome b2 or cytochrome b5 reductase/cytochrome b5) and iron-sulfur cluster proteins (adrenodoxin reductase/adrenodoxin) or by facultative one-electron acceptors such as benzoquinones (coenzyme... 

Cytochrome P-450 reductase (present in cell nuclear membranes) catalyzes reduction of the anthracyclines to semiquinone free radicals. These in turn reduce molecular 02, producing superoxide ions and hydrogen peroxide that mediate single strand scission of DNA (Figure 38.10). Tissues with ample superoxide dismutase (SOD) or glutathione peroxidase activity are protected.6 Tumors and the heart are generally low in SOD. In addition, cardiac tissue lacks catalase and thus cannot dispose of hydrogen peroxide. This may explain the cardiotoxicity of anthracyclines. 

Most reductases and dehydrogenases form neutral (blue) semi-quinones while most oxidases form anionic (red) semiquinones upon reduction by dithionite or anaerobic photoirradiation in the presence of EDTA (I). 

Fio. 13. Yeast glutathione reductase, semiquinone anion production from the 2-electron reduced form. Curve 1, oxidized enzyme, anaerobic conditions, pH 7.6 curve 2, 1 min after the addition of 1 equivalent of NADPH curve 3, 22 hr later curve 4, 1 hr after the addition of 10 equivalents of NADP curve 5, 235 hr later curve 6, 185 hr after the addition of 5 equivalents of NADPH and ciirve 7, 36 min after opening to air. 

The formation of a blue (neutral) semiquinone in high yield upon irradiation of thioredoxin reductase in the presence of a large excess of EDTA is shown in Fig. 3a. The semiquinone is further reduced to FADH at an even slower rate with maximal semiquinone formation at 4 hr. In contrast to this very slow semiquinone production, enzyme reduced by NADPH in the dark and subsequently exposed to light is rapidly converted to the semiquinone. The rate depends on the amount of NADPH used in the reduction with 0.5 mole NADPH per FAD the half-time is less than 0.5 min, with 2.0 moles NADPH per FAD the half-time is about 2 min. The rate of free radical production (EPR) exactly parallels the rate of increase in absorbance at 580 nm. The exact spectral characteristics of the semiquinone depend on the state of oxidation of the disulfide-dithiol. In the dithiol form the maximum is at 578 nm while in the disulfide form the maximum is at 588 nm. That the spectral properties are determined by the redox state of the disulfide is indicated by three findings. If semiquinone is produced by irradiation following reduction of the enzyme by 0.5 mole/FAD, the maximum is at 588 nm, while if the semiquinone is formed following reduction by 2.0 moles/FAD, the maximum is at 578 nm. Oxidation of enzyme irradiated in the presence of excess EDTA for various lengths of time requires ferricyanide stoichiometric with the ob-... 

Redox potentials have been determined for each of the steps of reduction of the trypsin-solubilized reductase (403) step 1, one electron consumed, Eo = —109 mV step 2, two electrons consumed. Eg = —276 mV and step 3, one electron consumed. Eg = —371 mV at pH 7.0, 26°. As expected, the redox potential of step 3 is more negative than the potential of the NADPH-NADP+ couple and was determined from the dithio-nite titration. The overall potentiometric—spectrophotometric titration curves could be very closely fitted with a computer-generated curve based on the assumptions of four one-electron reduction steps and octinction coefficients of 4.9 and 4.5 mM cm for the semiquinones, FliH and rijH the Eg values assumed for steps 2 and 3 were —270 and —290 mV. The precise fit was very sensitive to all of the assumptions (40 ) ... 

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. 

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