5-Deazaflavin radicals

Figure 7. Structure and decay of 5-deazaflavin radicals. The 5-protonated tautomer, which would be analogous to the biologically essential blue flavo-semiquinone, is not formed, and disproportionation, which is characteristic for the flavin system, does not happen.

Meanwhile we can show (50) that the deazaflavin radical generated by photodissociation of the dimer as shown in Figure 7 can be utilized for selective reduction of flavodoxin radical. With the aid of this le -transfer method Scherings, Haaker, and Veeger (51) were able to show that flavodoxin is the actual electron donor for the Azotobacter nitrogen-ase system and that N2 reduction can be maintained in the light by an artificial chain system of the reactant sequence EDTA-deazaflavin—flavodoxin—nitrogenase. Hence, Azotobacter flavodoxin is actually a carrier... 

Apart from the nuclear bromination observed (Section 2.15.13.1) in the attempted radical bromination of a side-chain methyl group leading to (396), which may or may not have involved radical intermediates, the only other reaction of interest in this section is a light-induced reduction of certain hydroxypyrido[3,4-f)]pyrazines or their 0x0 tautomers analogous to that well-known in the pteridine field (63JCS5156). Related one-electron reduction products of laser photolysis experiments with 1 -deazaflavins have been described (79MI21502). 

However, these experiments may not have established a mechanism for natural flavoprotein catalysis because the properties of 5-deazaflavins resemble those of NAD+ more than of flavins.239 Their oxidation-reduction potentials are low, they do not form stable free radicals, and their reduced forms don t react readily with 02. Nevertheless, for an acyl-CoA dehydrogenase the rate of reaction of the deazaflavin is almost as fast as that of natural FAD.238 For these enzymes a hydride ion transfer from the (3 CH (reaction type D of Table 15-1) is made easy by removal of the a-H of the acyl-CoA to form an enolate anion intermediate. 

Hence, the deazaflavin system can be forced to form radicals, but they are kinetically and thermodynamically unstable. Their structure is analogous to the biologically inessential N(l)-blocked red flavin radicals observed only in nonelectron transfer flavoproteins, in agreement with the fact that N( 1)-blockade shifts the flavin system ready for 2e -transfer only. The deazaflavoprotein radical, which has been observed by Hersh... 

The anaerobic reduction of the trinuclear copper center for ascorbate oxidase with different substrates presents a distinct picture. The reaction with reductate is monophasic with a unimolecular rate constant of 100 sec (18), independent of pH. Rapid freeze-quench EPR experiments indicate that the type-2 EPR signal vanishes more slowly 18). The pulse radiolysis studies of the radicals of lumiflavin, deazaflavin, CO2 ", and MV at pH 7.0 129,130) showed a biphasic behavior with an initial, faster reaction k = 97-127 sec " ) and a final, slower reaction k 2 sec" ) 129). Different results have been obtained by Farver and Pecht 130) with CO2 " as a substrate. They found a triphasic reaction with unimolecular rate constants k = 201 sec S 2 = 20 sec", and ks = 2.3 sec. The first constant is twice that in a study by Kyritsis et al. 129), whereas the third constant is identical. The second constant was not observed in the study. 

Both GcpE and LytB must be associated with a reducing system to convert the oxidized [Fc4S4] + cluster into the active reduced [Fc4S4] + form (Fig. 3), which can be performed with the isolated enzymes from E. coli either by the biologic system flavodoxin/flavodoxin reductase/NADPH, by the semiquinone radical of 5-deazaflavin, or by dithionite. In cyanobacteria, and in plant chloroplasts where flavodoxin is absent, this reducing... 

Figure 15 Cleavage of S-adenosyl-L-methionine by enzymes within the radicai SAM superfamiiy. The reductive cleavage reaction generates a 5 -deoxyadenosyl 5 -radical and L-methionine, a spectator in the reaction. The physiological electron donor is the flavodoxin/flavodoxin reductase reducing system, with electrons deriving ultimately from NADPH. Artificial electron donors such as sodium dithionite and 5-deazaflavin plus light can also effect reduction in in vitro activity determinations.

We have now from single turnover experiments direct evidence for an electron transfer from the reduced cluster to AdoMet as an elementary process involved in glycyl radical formation [40]. The pi protein could be reduced to the EPR-active form containing the [4Fe-4S]+ center by deazaflavin and then reacted with AdoMet in the dark, i.e. in the absence of a continuous electron flow. Oxidation of the cluster could be monitored by EPR spectroscopy and an assay for methionine served to measure the one-electron reduction of AdoMet. It appeared clear that one equivalent of methionine was formed at the expense of one equivalent of the reduced... 

DNA photolyases, which use the energy of blue light to split pyrimidine dimers formed by UV irradiation of DNA, provide other examples of large and variable shifts in the absorption spectmm of a bound chromophore. These enzymes contain a bound pterin (methylenetetrahydrofolate, MTHF) or deazaflavin, which serves to absorb light and transfer energy to a flavin radical in the active site [81]. The absorption maximum of MTHF occurs at 360 nm in solution, but ranges from 377 to 415 nm in the enzymes from different organisms [82]. 

Another milestone in flavin research was the characterization of the flavo-semiquinones, the first example of a stable flavin radical. In addition to riboflavin, FMN, and FAD, a number of flavin analogs with biological activities have been found in micro-organisms and plants. Among these, the coenzyme factor F420 isolated from methanogenic bacteria should be mentioned, which has 5-deazaflavin as its chromophore. 

Nowadays, a characteristically modified flavin, 5-deazaflavin (33,142), has gained much interest in flavin enzymology because it can replace natural flavin at many enzyme sites. This derivative, however, is no quinoid system and its radical has no thermodynamic stability. It is, therefore, a mutilated flavin retaining the 2e"-transfer (i.e. nicotinamidelike) properties only (cf. Table 2). The thia-analog, however, is an O2-stable dihydro derivative, which exhibits a stable radical, but no reversibly reducible oxidized form. Hence, it is also a mutilated flavin, but one which retains the e"-transferase activity only. 

Photodehydrogenation is entirely independent of the stability of the potentially intermediate radicals. 5-Deazaflavins, which are known to resist le -uptake, undergo analogous photoreductions as smoothly as flavins (JJ). 

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