Structure and properties of 5-deazaflavin radicals as compared to natural flavosemiquinones.

In order to gain more insight into flavin radicals, on which the selection of 1e-, and 2e- -oxireduction modes in flavoproteins depends, we have investigated structure, spectral properties and decay mode of molecular species occurring in the half-reduced 5-deazaflavin "model" system by flash photolysis and pulse radiolysis. (1) Enforced 1e- -reduction of 5-deazaflavin yields the short-lived red-colored 1-HdFl, which is a strong reductant. In the absence of any electron acceptor, this radical decays by 1,5-prototropy (see below) and dismutation, which is rapidly reversed upon illumination. Competing with this photo-comproportionation, irreversible formation of the photo-stable sigma-dimmer (HdFl)2, covalently linked via C(5), is observed, which becomes prevalent under prolonged illumination. (2) Enforced 1e- -abstraction from 1,5-dihydro-5-deazaflavin yields the tautomeric 5-HdFl, which is a mild oxidant and is transparent at lambda 480 nm. Prototropy 5-HdFl in equilibrium or formed from 1-HdFl can be rate-determining in 5-deazaflavin redox reactions. Hence, the radical state in the 5-deazaflavin system does not mediate double 1e- -oxidoreduction as do natural flavosemiquinones. Instead, 5-deazaflavin flavors nucleophilic substrate addition (carbanion transfer) and formation of intermediate sigma-adducts in (photo)reductions even over the extent observed with natural flavin. This confirms the description of 5-deazaflavin as a "flavin-shaped nicotinamide derivative". It explains at the same time the mechanism of 5-deazaflavin acting as a mild and yet potent photosensitizer in 1e- -reductions of biological redox systems. (3) It is shown that replacement of N(5) by CH in the flavin nucleus also leads to the disappearance of the known action-pK in the photoreduction, which confirms the assignment of the latter pK in the natural flavin system to 5-protonation of the excited flavin triplet. From these model studies the following biological conclusions can be confirmed: The tautomer equilibrium of natural flavin semiquinones is diffusion-controlled and regulated thermodynamically: 5-HFl in equilibrium or formed from 1-HFl, while in flavo-proteins the same equilibrium is regulated by regiospecific H-bridges from the apoprotein, which thus decides between 1e- - (stable 5-HFl) and 2e- -reaction (unstable 1-HFl) modes.