pK Values of the Cofactor Tune the Redox Regime of Flavoenzymes.

Across the diverse family of flavoenzymes, the isoalloxazine cofactor was found to display extremely diverse redox properties, both with respect to the absolute value of the potential regime wherein it operates and to its redox cooperativity, that is, the relative positioning of its individual 1-electron transitions. Taking together electrochemical data and 3D structural information reported for selected representatives of the flavoenzyme family, we assessed the contribution of pK value modifications at the three protonatable nitrogens of the isoalloxazine moiety. While the absolute value of the redox regime appears only weakly dependent on such pK modifications, the diversity of redox cooperativity is readily rationalized by (protein-induced) stabilization/destabilization of the proton primarily on N5 and to lesser degrees on N1 and N3. The mathematical formalism underlying the interdependence of pK values and redox midpoint potentials is subsequently extended to representatives of the family featuring extremely positive redox cooperativity (i.e., the electron bi/confurcating flavoenzymes). Observed structural idiosyncrasies of these cases were found to rationalize the extremely strong inversion (ΔE ≪ -800 mV) of 1-electron midpoint potentials in the framework of this formalism.