Flavoproteins are a ubiquitous class of redox enzymes that participate in a host of biological processes including O2 activation, DNA repair, aromatic hydroxylation, and magnetoreception.  Their diverse utility stems from the flavin cofactor's ability to use light or thermodynamic driving forces to transfer one or two electrons and/or a hydride over redox potentials spanning ~600mV. These properties are modulated by specific interactions between the flavin and the surrounding protein matrix.  In this work we aim to design non-natural flavoproteins for electron transfer chemistry using engineering principles learned from natural proteins.   We utilize a robust, highly controlled model system: tetra-helical protein maquettes, to which we covalently coupled flavin analogues to an engineered cysteine.   Thus far, we have used strategic amino acid substitutions to alter the environment surrounding the flavin and tune its redox potential by ~200mV.  We have also constructed a maquette that shows light activated electron transfer between a flavin and bis-his ligated Heme B.  In order to stabilize the charge separated state, we have added a redox active tryptophan to form a triad.  The biophysical and photochemical characterization of these designs will be presented.  These results not only help us understand the natural light/redox active flavoproteins but also bring us closer to realizing fully synthetic flavin enzymes for novel catalysis.

Engineering manmade flavoproteins for electron transfer and beyond

247th National Meeting (2014)

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