Modulation of function in a minimalist heme-binding membrane protein.
De novo designed heme-binding proteins have been used successfully to recapitulate features of natural hemoproteins. This approach has now been extended to membrane-soluble model proteins. Our group designed a functional hemoprotein, ME1, by engineering a bishistidine binding site into a natural membrane protein, glycophorin A (Cordova et al. in J Am Chem Soc 129:512-518, 2007). ME1 binds iron(III) protoporphyrin IX with submicromolar affinity, has a redox potential of -128 mV, and displays peroxidase activity. Here, we show the effect of aromatic residues in modulating the redox potential in the context of a membrane-soluble model system. We designed aromatic interactions with the heme through a single-point mutant, G25F, in which a phenylalanine is designed to dock against the porphyrin ring. This mutation results in roughly tenfold tighter binding to iron(III) protoporphyrin IX (K(d,app) = 6.5 × 10(-8) M), and lowers the redox potential of the cofactor to -172 mV. This work demonstrates that specific design features aimed at controlling the properties of bound cofactors can be introduced in a minimalist membrane hemoprotein model. The ability to modulate the redox potential of cofactors embedded in artificial membrane proteins is crucial for the design of electron transfer chains across membranes in functional photosynthetic devices.
Department of Chemistry and Biochemistry, ASU, Tempe, AZ 85287, USA.
Sandip
S
Shinde
Jeanine M
JM
Cordova
Brian W
BW
Woodrum
Giovanna
G
Ghirlanda
D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000081062:Heme-Binding Proteins; D006420:Hemeproteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D009154:Mutation; D010084:Oxidation-Reduction; D011199:Potentiometry; D011524:Protoporphyrins / Q000737:chemistry / Q000378:metabolism
D002352:Carrier Proteins; D000081062:Heme-Binding Proteins; D006420:Hemeproteins; D008565:Membrane Proteins; D011524:Protoporphyrins; C448299:iron protoporphyrin IX