Design of a Five-Coordinate Heme Protein Maquette:  A Spectroscopic Model of Deoxymyoglobin

Abstract

The substitution of 1-methyl-l-histidine for the histidine heme ligands in a de novo designed four-alpha-helix bundle scaffold results in conversion of a six-coordinate cytochrome maquette into a self-assembled five-coordinate mono-(1-methyl-histidine)-ligated heme as an initial maquette for the dioxygen carrier protein myoglobin. UV-vis, magnetic circular dichroism, and resonance Raman spectroscopies demonstrate the presence of five-coordinate mono-(1-methyl-histidine) ligated ferrous heme spectroscopically similar to deoxymyoglobin. Thermodynamic analysis of the ferric and ferrous heme dissociation constants indicates greater destabilization of the ferric state than the ferrous state. The ferrous heme protein reacts with carbon monoxide to form a (1-methyl-histidine)-Fe(II)(heme)-CO complex; however, reaction with dioxygen leads to autoxidation and ferric heme dissociation. These results indicate that negative protein design can be used to generate a five-coordinate heme within a maquette scaffold.

Computational Design and Elaboration of a de Novo Heterotetrameric α-Helical Protein That Selectively Binds an Emissive Abiological (Porphinato)zinc Chromophore

“…Such estimates are approximate and nonrigorous thermodynamically due to the poor solubility of the cofactor and the presence of detergent in the samples. Notwithstanding, this analysis provides an empirical description of the binding process that demonstrates: 1) a high cooperativity to the assembly process (the first cofactor is bound weakly, while the second is bound tightly); 2) the mean overall dissociation constant, (K 1 K 2 ) 1/2 = 0.28, is in the sub-micromolar range (and consistent with values found for a five-coordinate heme-protein maquette) 7,61. Ratiometric titrations (Job plot analysis) involving the two peptides ( A His : B Thr : 1:0 → 1:1 → 0:1) evince a maximal (DPP)Zn binding efficiency at 1:1 A His : B Thr (Figure 4, inset), consistent with the targeted structure of the complex.…”

Protein Design: Toward Functional Metalloenzymes

“…This particular modification decreased the ferric heme affinity dramatically, resulting in a well-defined ferrous heme center that binds CO but not O 2 . 264 The construct containing two 3-methyl-histidines, [Δ H 3m] 2 , was used to examine the roles of the side chains on heme a , which differs from heme b by the presence of a C-2 hydroxyethylfarnesyl group and a C-8 formyl group. 265 The affinity, spectroscopy, and electrochemistry were compared between heme b and a heme a mimic, diacetyldeuterioporphyrin IX (DADPIX).…”

Controlling and Fine Tuning the Physical Properties of Two Identical Metal Coordination Sites in De Novo Designed Three Stranded Coiled Coil Peptides

“…The Dutton and Gibney groups have used four-helix bundles as maquettes to study heme containing proteins 6–9. Gibney and coworkers also used the maquette motifs to develop a prototype ferredoxin maquette, a peptide-based synthetic analogue of natural [4Fe-4S] 2+/+ proteins 10.…”