5-deazariboflavin semiquinone

A flash photolysis method has been developed that prepares the MoVI-Fe11 state and thus allows the rate constants k3 and k 3 to be measured. Solutions containing 5-deazariboflavin, semicarbazide, and sulfite oxidase are subjected to 555 nm flash photolysis. The deazariboflavin is excited to a triplet state, which is then reduced by semicarbazide to form the 5-deazariboflavin semiquinone radical. This radical is then rapidly oxidized back to its parent species through the one-electron reduction of sulfite oxidase. 

The rate of intramolecular electron transfer from the flavin to the heme in PCMH has been measured by laser flash photolysis using 5-deazariboflavin semiquinone radical as a 1-electron reductant (Bhat-... 

Deazariboflavin semiquinone (DRFLH ) had been shown to exhibit low reactivity (/ 10 s ) with the heme of CCP (34) thus, DRFLH was... 

Recently we carried out kinetic studies with Hildenborough and Miyazaki cytochrome c3 using deazariboflavin semiquinone (dRf ), MV +, and propylene diquat (PDQ +), produced by laser flash photolysis, as reductants (37). Initially, all three reactions were accurately second order, consistent with all hemes being reduced with the same rate constant or with a single site reduced, followed by fast intramolecular electron transfer to reduce the remaining three hemes. However, by measuring reduction kinetics with cytochrome c3 poised at different extents of reduction, the kinetics of reduction of individual hemes could be resolved. Thus, reduction of cytochrome c3 in approximately 5% steps and application of the known macroscopic redox potentials (see previous section) enabled calculation of the concentration of each heme (c.) at each stage of reduction. The plot of kohs versus percent reduction can thereby be fitted to solve for the rate constant for each heme (kt) ... 

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). 

Laser flash photolysis time-resolved spectrophotometry, utilizing deazariboflavin-EDTA as a photochemical reductant, has been used with this system in order to characterize the initial step in the ET mechanism. Figure 3 shows examples of the type of data obtained in these studies. In the top panel, a transient is shown [54] that was obtained at 507 nm in 100 mM phosphate buffer, pH 7.0, containing 35 pM Fd, and in the middle panel, 10.3 pM FNR has been added to the solution prior to photolysis. This wavelength corresponds to an isosbestic point for the FAD cofactor of the reductase, and thus the absorbance change monitors the oxidation state of the [2Fe-2S] cluster of Fd (and also the formation and decay of the dRfH species). As is evident, immediately after the laser flash there is a rapid rise in absorbance due to dRfH formation. This is followed by a sharp absorbance decrease corresponding to Fd reduction and dRfH oxidation. The subsequent slow increase in absorption shown in the middle panel is a consequence of Fd reoxidation that is due to electron transfer to FNR. The latter is confirmed by measurement at 610 nm (bottom panel), a wavelength which monitors FAD neutral semiquinone formation the rate constant obtained from the 610 nm absorbance rise is the same as that obtained from the slow absorbance increase at 507 nm, consistent with this interpretation. 

Laser flash photolysis studies using the deazariboflavin system [80, 81] have shown that pyruvate binding also exerts a strong influence on intramolecular ET. In the one-electron reduced enzyme, ET from the FMN semiquinone to the oxidized heme can be observed only in the presence of pyruvate for this reaction, k j = 500 s. When the enzyme is completely reduced by stoichiometric addition of lactate prior to laser photolysis with dRf alone, pyruvate binding inhibits ET from fully reduced flavin to oxidized heme. This reaction has an observed /cet = 2000 s in the absence of pyruvate [81]. Similar values for these two rate constants have been obtained by temperature-jump measurements [82]. 

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