Functionalized de novo designed proteins: Mechanism of proton coupling to oxidation/reduction in heme protein maquettes

Proton exchange with aqueous media coupled to heme oxidation/reduction is c ommonly seen but not understood in natural cytochromes. Our synthetic tetra helix bundle heme protein maquettes successfully reproduce natural proton c oupling to heme oxidation/reduction. Potentiometry reveals major pK shifts from 4.2 to 7.0 and from 9.3 to 10.3 in the maquette-associated acid/base g roup(s) upon heme reduction. Consequently, a 210 mV decrease in the heme re dox potential is observed between the two extremes of pH. Potentiometry wit h resonance Raman and FTIR spectroscopy performed over a wide pH range stro ngly implicates glutamate side chains as the source of proton coupling belo w pH 8.0, whereas lysine side chains are suggested above pH 8.0. Remarkably , the pK values of several glutamates in the maquette are elevated from the ir solution value (4.4) to values as high as 7.0. It is suggested that thes e glutamates are recruited into the interior of the bundle as part of a str uctural rearrangement that occurs upon heme binding. Glutamate to glutamine variants of the prototype protein demonstrate that removal of the glutamat e closest to the heme diminishes but does not abolish proton exchange. It i s necessary to remove additional glutamates before pH-independent heme oxid ation/reduction profiles are achieved. The mechanism of redox-linked proton coupling appears to be rooted in distributed partial charge compensation, the magnitude of which is governed by the dielectric distance between the f erric heme and acid/base side chains. A similar mechanism is likely to exis t in native redox proteins which undergo charge change upon cofactor oxidat ion/reduction.