Aromatic side chain-porphyrin interactions in designed hemoproteins

Aromatic amino acid side chains are commonly observed to interact with the heme cofactors of natural hemoproteins. These interactions are of the types previously identified for pairs or groups of aromatic amino acid side chai ns in proteins: offset pi-stacking and T-stacking (an edge-to-face arrangem ent). To evaluate how such interactions may influence structural stability of hemoproteins, we synthesized peptide-sandwiched mesohemes (PSMs) 2 and 3 in which the alanine-4 (Ala-4) residues in 1 have been replaced by phenyla lanine (Phe) and tryptophan (Trp), respectively. The Co(III) analogues of 1 , 2, and 3 (l-Co, 2-Co, and 3-Co, respectively) were also prepared. Histidi ne (His)-to-iron coordination in 1 had previously been shown to induce heli cal conformations in the peptides (helix content similar to 50% at 8 degree s C). Molecular modeling studies suggested that Trp, but not Phe, could eng age in edge-to-face interactions with the porphyrin if the peptides are ful ly helical. Replacing Ala-4 with Trp, bur not with Phe, was thus predicted to Favor enhanced peptide helix content. Circular dichroism spectra are con sistent with significantly increased helix content in 3 relative to i, but not in 2. Hydrogen-deuterium (H/D) exchange rates determined by electrospra y ionization mass spectrometry, however, decrease in the order 1 much great er than 2 > 3, while pH titrations reveal that the stability of the model p rotein folds decreases in the order 3 > 2 much greater than 1. Furthermore, H-1 NMR spectra of 2-Co and 3-Co indicate that the aromatic side chains in each compound are oriented within the shielding region of the porphyrin ri ng. Two-dimensional NOE and chemical shift data show that the helices in 3- Co are more highly organized than in 1-Co and span nearly the entire peptid e sequence, while in 2-Co shorter helices of intermediate stability run bet ween Phe-4 and Ala-13. The combined results indicate that aromatic side cha in-porphyrin interactions in 2 and 3 stabilize their respective model prote in folds, and suggest a similar role for the corresponding interactions in natural hemoproteins. Finally, the chemical shift patterns of the Trp side chains in 3-Co, the different effects of Phe and Trp on peptide architectur e, and the pattern of chemical shifts exhibited by the alpha-NH and alpha-C H hydrogens in all three Co(III) PSMs demonstrate that the solution structu res of these designed hemoproteins are similar to those predicted in molecu lar modeling studies.