The effects of various mechanisms of metalloporphyrin reduction potential modulation were investigated experimentally using a robust, well-characterized heme protein maquette, synthetic protein scaffold H10A24 [¿CH(3)()CONH-CGGGELWKL.HEELLKK.FEELLKL.AEERLKK. L-CONH(2)()¿(2)](2). Removal of the iron porphyrin macrocycle from the high dielectric aqueous environment and sequestration within the hydrophobic core of the H10A24 maquette raises the equilibrium reduction midpoint potential by 36-138 mV depending on the hydrophobicity of the metalloporphyrin structure. By incorporating various natural and synthetic metalloporphyrins into a single protein scaffold, we demonstrate a 300-mV range in reduction potential modulation due to the electron-donating/withdrawing character of the peripheral macrocycle substituents. Solution pH is used to modulate the metalloporphyrin reduction potential by 160 mV, regardless of the macrocycle architecture, by controlling the protonation state of the glutamate involved in partial charge compensation of the ferric heme. Attempts to control the reduction potential by inserting charged amino acids into the hydrophobic core at close proximity to the metalloporphyrin lead to varied success, with H10A24-L13E lowering the E(m8.5) by 40 mV, H10A24-E11Q raising it by 50 mV, and H10A24-L13R remaining surprisingly unaltered. Modifying the charge of the adjacent metalloporphyrin, +1 for iron(III) protoporphyrin IX or neutral for zinc(II) protoporphyrin IX resulted in a loss of 70 mV [Fe(III)PPIX](+) - [Fe(III)PPIX](+) interaction observed in maquettes. Using these factors in combination, we illustrate a 435-mV variation of the metalloporphyrin reduction midpoint potential in a simple heme maquette relative to the about 800-mV range observed for natural cytochromes. Comparison between the reduction potentials of the heme maquettes and other de novo designed heme proteins reveals global trends in the E(m) values of synthetic cytochromes.

We report the construction of a synthetic f lavo-heme protein that incorporates two major physiological activities of f lavoproteins: light activation of f lavin analogous to DNA photolyase and rapid intramolecular electron transfer between the f lavin and heme cofactors as in several oxidoreductases. The functional tetra-␣-helix protein comprises two 62-aa helix-loop-helix subunits. Each subunit contains a single cysteine to which f lavin (7-acetyl-10-methylisoalloxazine) is covalently attached and two histidines appropriately positioned for bis-his coordination of heme cofactors. Both f lavins and hemes are situated within the hydrophobic core of the protein. Intramolecular electron transfer from f lavosemiquinone generated by photoreduction from a sacrificial electron donor in solution was examined between protoporphyrin IX and 1-methyl-2-oxomesoheme XIII. Laser pulseactivated electron transfer from f lavin to meso heme occurs on a 100-ns time scale, with a favorable free energy of approximately ؊100 meV. Electron transfer from f lavin to the lower potential protoporphyrin IX, with an unfavorable free energy, can be induced after a lag phase under continuous light illumination. Thus, the supporting peptide matrix provides an excellent framework for the positioning of closely juxtaposed redox groups capable of facilitating intramolecular electron transfer and begins to clarify in a simplified and malleable system the natural engineering of f lavoproteins.Nature selects from a battery of redox active cofactors and assembles them within a protein matrix to facilitate essential functions such as substrate binding, electron transfer, energy conversion, and chemical catalysis. The cofactors often are juxtaposed closely within a single structure with the physical chemical properties adjusted by the protein environment to perform the desired function satisfactorily and with high fidelity. Studies aimed at understanding the molecular basis of catalytic fitness in enzymes often are confounded by the sheer complexity of natural proteins. It is our goal to uncover the engineering of natural oxidoreductase protein design by simplifying the problem and synthesizing the minimalistic protein structures that assemble working arrays of cofactors and reproduce native-like function. We call these working structures ''molecular maquettes.''To date there has been much success with this approach. The peptide scaffold of choice is a tetra-␣-helix-bundle that assembles and is stable over a wide range of conditions