To support the rapid expansion of global energy needs, new catalysts are required for more efficient conversion between electrical energy from renewable sources and chemical energy in the form of fuels. Structures incorporating cobalt porphyrins have shown promise in catalyzing O2 reduction, an important fuel cell reaction. Cobalt porphyrins can be introduced into a protein environment by substituting a heme iron for cobalt, expanding the functionality of metalloproteins through rational design. The goal of this study is to engineer a novel di-heme protein with a stacked heme arrangement and explore its catalytic potential for O2-reduction upon substitution of the heme iron for cobalt. Carboxyl-terminus of Hsp70 interacting protein (CHIP) is a naturally occurring protein that has been chosen as a template by computer-based structural modeling. The structure of CHIP has recently been determined, revealing a pocket where two c-type hemes are predicted to stack upon protein dimerization. The CHIP template has been modified by the introduction of a Cys-X-X-Cys-His motif for heme c binding through the mutations F274C, E277C, and N278H. A K202H mutation has also been introduced to avoid steric clash with the heme. An N-terminal histidine tag has been replaced by a PelB signal sequence that will direct the protein to the periplasm of the cell where heme attachment to the Cys-X-X-Cys-His motif by the E. coli Ccm system can occur. The modified CHIP gene was cloned into a pRSETB-based expression vector using a the SLiCE (seamless ligation cloning extract) method, which utilizes extract from a RecA– strain of E. coli to perform in vitro recombination, eliminating the need for restriction enzymes or ligases. Expression and purification protocols are currently being developed, and the resulting samples will be tested for dimerization and for the presence of stacked hemes within the modified CHIP protein.

Engineering metalloproteins for fuel catalysis using SLiCE and CHIP template

253rd National Meeting (2017)

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