More than one third of all proteins are metalloproteins. They catalyze important reactions such as photosynthesis, nitrogen fixation and CO2 reduction. Metalloproteins are especially adept at catalyzing reactions requiring multiple electron and proton transfer. The key for high activity and selectivity, is the precise arrangement of chromophore, metal ions, cofactors and amino acids with optimal pKa and reduction potentials to serve as proton and electron relays for substrate activation. However, metalloproteins such as photosystem I and photosystem II (PSI and PSII) are extremely complex. This complexity has greatly hindered our understanding of the basic catalytic mechanism of reactions, and our ability to expand the functions of metalloproteins for their applications in synthetic biology. The goal in my laboratory is to rationally design small, simple metalloproteins which are capable to mimicking PSI and PSII, and catalyzing nitrogen fixation and CO2 reduction with much higher efficiency than natural enzymes. Toward this goal, we have made the following progress in recent years: 1. Genetic incorporation of unnatural amino acid probes to investigate photo-induced electron transfer in proteins. We have reported the genetic incorporation of the following unnatural amino acids (UAA): 3,5-dichlorotyrosine (Cl2Y), 3,5-difluorotyrosine-(F2Y), Cu(II) binding 3-pyrazoletyrosine(pyTyr), 4-fluoro-3-nitrophenylalanine (FNO2Phe). These UAAs have redox potentials spanning from -400 to 800 mV, which covers the whole range of biologically relevant redox potential. This new tool kit has allowed us to design superb photoactive enzymes and fluorescent sensors. 2. By using the genetic code expansion tools we have recently developed, we were able to design small photoactive protein which undergoes photo-induced electron transfer at a rate of 10 ps, similar to the rate of primary charge-separation process in PSI. Our recent results show that we are able to photo-catalytically drive efficient ferredoxin reduction, CO2 reduction, or Mn(II) oxidation, using rationally designed small photoactive proteins. This may allow us to design synthetic biological systems which have much higher photosynthesis efficiency than that of the native biological systems.

Metalloprotein design using genetic code expansion

256th National Meeting (2018)

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