Jeanine M. Cordova1, Pamela L. Noack1, Simon A. Hilcove1, James D. Lear2 and Giovanna Ghirlanda1,* 1Department of Chemistry and Biochemistry, Arizona State University, Tempe, AZ 85287-1604; 2Department of Biochemistry and Biophysics, University of Pennsylvania, Philadelphia Pennsylvania 19104

We have designed a functional model membrane protein by engineering a bis-Histidine heme-binding site into a natural membrane protein, glycophorin A (GpA), structurally characterized by the dimerization of a single transmembrane helix. Five amino acids out of the thirty-two residues comprising the transmembrane helix of GpA were mutated; the resulting protein, ME1, has been characterized in dodecyl phosphocholin (DPC) micelles by UV-vis, CD spectroscopy, gel electrophoresis and analytical ultracentrifugation. ME1 binds heme with sub-micromolar affinity, and maintains the highly helical secondary structure and dimeric oligomerization state of GpA. The ME1-Heme complex exhibits a redox potential of -128 „b 2 mV vs SHE, indicating that the heme resides in a hydrophobic environment and is well shielded from the aqueous phase. Most importantly, ME1 shows significant peroxidase activity; the catalytic efficiency, kcat/KM, for the hydrogen peroxide dependent oxidation of TMB (2,2',5,5'-tetramethyl-benzidine) is 2044 „b283 M-1 s-1 in the presence of ME1-heme, an improvement of two orders of magnitudes over the heme-catalyzed reaction, which has a second order rate constant of 10.31 M-1 s-1. This protein may provide a useful framework to investigate how the protein matrix tunes the cofactor properties in membrane proteins.

This work was supported by NSF CAREER Award 0449842 to G.G. and by an NSF IGERT fellowship to J.M.C.