Catalytic semiquinone

Therefore, the Rieske protein has to switch between the positional states during turnover. The following reaction scheme combines the movement of the catalytic domain of the Rieske protein with the redox-dependent stabilization of the intermediate semiquinone (Fig. 19) (42) ... 

P27 In this article, we report the results of studies that indicate that PMj 5 does indeed contain semiquinone-type radicals, and these radicals can initiate damage to DNA through a catalytic cycle involving ROS. (Adapted from Dellinger et al., 2001)... 

For dinuclear Cu complexes, several pathways are possible as summarized in Scheme 15 [182]. In addition, plausible alternatives involve mixed-valent Cu Cu species where only one of the Cu ions is directly involved in the electron transfer. The latter seems most hkely in cases where the substrate binds to only one of the two copper ions, and H2O2 may then form upon oxidation of the Cu Cu -semiquinone intermediate [195]. Different coordination modes of the DTBC substrate appear to be indeed possible, depending on the particular dicopper scaffold [133,196,197]. Unfortunately, detailed mechanistic studies are still quite scarce [198-203] and most proposed catalytic pathways are rather speculative. 

Enzyme-Bound Flavin Semiquinones which are not Catalytic... 

Each of the forms of ETF isolated from the different sources contain FAD as coenzyme and form an anionic semiquinone on one-electron reduction. Stopped-flow kinetic studies on the pig liver ETF showed the anionic flavin semiquinone to be formed at times faster than catalytic turnover and thus demonstrate the participation of the anionic FAD semiquinone as an intermediate in the acceptance of reducing equivalents from the dehydrogenase. These studies would also imply the intermediacy of the semiquinone form of the acyl CoA dehydrogenase which would have been expected to form a neutral flavin semiquinone at the time the studies of Hall and Lambeth were performed, however, no spectral evidence for its formation were found. Recent studies have shown that the binding of CoA analogs to the dehydrogenase results in the perturbation of the pKa of the FAD semiquinone such that an anionic (red) rather than the neutral (blue) semiquinone is formed. This perturbation was estimated to reduce the pKa by at least 2.5 units to a value of... 

The properties of the semiquinone from of the ETF isolated from the methylotrophic bacterium resemble those of the bacterial flavodoxins with the exception that flavodoxins form neutral semiquinones whereas this ETF forms an anionic semiquinone. Nearly quantitative anionic semiquinone formation is observed either in the presence of excess dithionite or when excess trimethylamine and a catalytic amount of trimethylamine dehydrogenase are added. Of interest is the apparent stability of the anionic semiquinone towards oxidation by O2 but not to oxidizing agents such as ferricyanide. This appears to be the first example of an air-stable protein-bound anionic flavin semiquinone. Future studies on the factors involved in imparting this resistance to O2 oxidation by the apoprotein are looked forward to with great interest. 

Enzyme-Bound Flavin Semiquinone which are not Catalytic Intermediates... 

A considerable amount of information regarding flavin semiquinone reactivity as well as the environment of the bound flavin coenzyme has accumulated over the years from studies of flavoenzyme systems which produce semiquinones either on photochemical reduction or upon reduction by one electron equivalent of dithionite, but which do not form a detectable semiquinone intermediate during catalytic turnover. For example, the correlation of anionic semiquinone formation and the ability to bind sulfite at the N(5) position in a number of flavoenzyme... 

Semiquinone formation is undoubtedly of great significance in physiological processes. Thus it has been found64 that whereas diamino-durene. increases the respiration of erythrocytes to about the same extent as methylene blue, tetramethyldiaminodurene has no catalytic effect at all. 

The mechanism of the catalytic reaction proved indeed to be very different from that found for [Cu2([22]py4pz)( r-0H)](C104)3 H20. Thus, in the first step of the reaction, a stoichiometric oxidation of catechol by the dicopper(II) complex takes place however, only one electron is transferred in this stoichiometric reaction, resulting in the formation of a semiquinone radical and a mixed-valence Cu"Cu species. Interestingly, the dicopper(II) complex was found to be essentially dinuclear in solution nevertheless, only one of the two copper(II) ions was found to participate in the redox process, whereas the second one played a purely structural... 

The central ring of 1-deazaflavins remains a pyrazine in X, a di-hydropyrazine in the two-electron-reduced form, XI, and continues to dominate the chemistry with oxygen. Like the parent riboflavins, and unlike the 5-deazaflavins, the dihydro- 1-deaza system, XI, is reoxidized by 02 in a fraction of a second in air-saturated solutions (Table II) the semiquinone is accessible and 1-deazaFAD enzymes show full catalytic competence with flavoprotein dehydrogenases and oxidases (24). Turnover numbers vary from about 1% to 100% that of cognate FAD-enzymes but this variation reflects the -280 mV vs. —200 mV E° values, respectively, for 1-deazariboflavin vs. riboflavin. The redox steps may or may not limit Vmax with a given enzyme (15, 24). 

The lack of reactivity of the semiquinone per se with either thioredoxin or NADPH shows that it cannot be involved in catalysis. The rapid production of semiquinone by irradiation of partially reduced enzyme is a light-activated disproportionation since it is totally dependent upon the presence of some oxidized enzyme. Enzyme fully reduced by dithionite forms no semiquinone, while enzyme partially reduced by dithionite rapidly forms semiquinone upon irradiation. Furthermore, the light-activated disproportionation of enzyme first reduced with NADPH results in the reduction of NADP. Thus, FAD catalyzes the disproportionation in keeping with the known photosensitizing nature of free flavins. This reaction is reversed slowly (half-time ca. 150 min 25°) in the dark. The semiquinone is rapidly reoxidized by oxygen to yield an enzyme with unaltered spectral and catalytic properties (58). Similar reactions have been very briefly reported for lipoamide dehydrogenase the dark reverse reaction is comparatively rapid, being complete in 30 min (16S). 

The vast majority of flavoenzymes catalyze oxidation-reduction reactions in which one substrate becomes oxidized and a second substrate becomes reduced and the isoalloxazine ring of the flavin prosthetic group (Figure 1) serves as a temporary repository for the substrate-derived electrons. The catalytic reaction can be broken conveniently into two steps, a reductive half reaction (from the viewpoint of the flavin) and an oxidative half reaction. The flavin ring has great utility as a redox cofactor since it has the ability to exist as a stable semiquinone radical. Thus, a flavoenzyme can oxidize an organic substrate such as lactate by removal of two electrons and transfer them as a pair to a 2-electron acceptor such as molecular oxygen, or individually to a 1-electron acceptor such as a cytochrome. 

During catalytic turnover, NADPH reduces FAD and the FAD subsequently reduces FMN by two electrons with the latter eyeling between the hydroquinone and semiquinone states while carrying out the 1-electron reduction of the P450 heme iron (Masters et al., 1966 Backes and Reker-Backes, 1988). The midpoint redox potentials of the FAD are El(,x/sq n290 mV and fi365 mV and of the FMN are El(,x/sq nl lOmV and... 

Wamcke, K., Brooks, H. B., Babcock, G. T, Davidson, V. L., and McCracken, J. L., 1993, The Nitrogen atom of substrate methylamine is incorporated into the tryptophan tryptophyl-semiquinone catalytic intermediate in methylamine dehydrogenase, J. Am. Chem. Soc. 115 6464n6465. 

It will be seen from Figure 13 that copper is not invoked for the catalytic steps of the reductive half cycle and an unresolved issue is whether the copper-free form of amine oxidase can catalyse the reactions leading to the aminoquinol form of the TPQ cofactor and release of product aldehyde. For the lentil seedling amine oxidase (Rinaldi et al., 1989), it has been reported that the copper-free form can catalyse the reactions of the reductive half cycle but that the presence of copper is required for the TPQ semiquinone species to be observed (Bellelli et al., 1985). Further work is needed to establish fully the catalytic properties of apo-forms and metal substi-tuted-forms of amine oxidases. 

Electron flow through flavocytochrome bz has been extensively studied in both the S. cerevisiae (Tegoni et al., 1998 Daff et al., 1996a Chapman et al., 1994 Pompon, 1980) and H. anomala (CapeillEre-Blandin et al., 1975) enzymes. The catalytic cycle is shown in Figure 3. Firstly, the flavin is reduced by L-lactate a carbanion mechanism has been proposed for this redox step (Lederer, 1991). Complete (two-electron) reduction of the flavin is followed by intra-molecular electron transfer from fully-reduced flavin to heme, generating flavin semiquinone and reduced heme (Daff et al.. 

FIGURE 3. The Catalytic Cycle for Flavocytochrome 62 F, flavin H, heme Cyt c, cytochrome c. Electrons are shown as hlack dots and are used to indicate the two-electron reduced flavin (hydroquinone), F with two dots one-electron reduced flavin (semiquinone), F with one dot reduced heme, H with one dot reduced cytochrome c, Cyt c with one dot. The rate constants shown are for S. cerevisiae flavocytochrome hi at 25 C, pH 7.5, I = O.IOM. The whole catalytic cycle turns over at approximately 100 s ". The details of the cycle are described in the main text. 

Whiehever mechanism operates, it is clear that the rate of reduetion of the flavin group is totally limited by the cleavage of the aC-H bond sinee the deuterium kinetic isotope effect for this step is around 8 (Miles et al., 1992 Pompon et al., 1980). However, in flavocytochrome 2 the rate of flavin reduetion is some 6-fold faster than the overall steady-state turnover rate (Daff et al., 1996a). As a consequence the flavin reduction step eontributes little to the rate limitation of the overall catalytic cycle (Figure 3). In faet it is eleetron transfer from flavin-semiquinone to b2 -heme that is the major rate-determining step and this is discussed in the following seetion. 

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