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Oxygen reactivity of the biferrous site in the de novo designed four helix bundle peptide DFsc: nature of the "intermediate" and reaction mechanism.

ABSTRACT: The DFsc and DFscE11D de novo designed protein scaffolds support biomimetic diiron cofactor sites that react with dioxygen forming a 520 nm "intermediate" species with an apparent pseudo-first-order formation rate constant of 2.2 and 4.8 s-1, respectively. Resonance Raman spectroscopy shows that this absorption feature is due to a phenolate-to-ferric charge transfer transition arising from a single tyrosine residue coordinating terminally to one of the ferric ions in the site. Phenol coordination could provide a proton to promote rapid loss of a putative peroxo species.

SUBMITTER: Calhoun JR

PROVIDER: S-EPMC3810014 | biostudies-literature | 2008 Jul

REPOSITORIES: biostudies-literature

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Oxygen reactivity of the biferrous site in the de novo designed four helix bundle peptide DFsc: nature of the "intermediate" and reaction mechanism.

Calhoun Jennifer R JR Bell Caleb B CB Smith Thomas J TJ Thamann Thomas J TJ DeGrado William F WF Solomon Edward I EI

Journal of the American Chemical Society 20080624 29

The DFsc and DFscE11D de novo designed protein scaffolds support biomimetic diiron cofactor sites that react with dioxygen forming a 520 nm "intermediate" species with an apparent pseudo-first-order formation rate constant of 2.2 and 4.8 s-1, respectively. Resonance Raman spectroscopy shows that this absorption feature is due to a phenolate-to-ferric charge transfer transition arising from a single tyrosine residue coordinating terminally to one of the ferric ions in the site. Phenol coordinatio ...[more]

PMID: 18572936

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Project description:De novo protein design represents an attractive approach for testing and extending our understanding of metalloprotein structure and function. Here, we describe our work on the design of DF (Due Ferri or two-iron in Italian), a minimalist model for the active sites of much larger and more complex natural diiron and dimanganese proteins. In nature, diiron and dimanganese proteins protypically bind their ions in 4-Glu, 2-His environments, and they catalyze diverse reactions, ranging from hydrolysis, to O2-dependent chemistry, to decarbonylation of aldehydes. In the design of DF, the position of each atom-including the backbone, the first-shell ligands, the second-shell hydrogen-bonded groups, and the well-packed hydrophobic core-was bespoke using precise mathematical equations and chemical principles. The first member of the DF family was designed to be of minimal size and complexity and yet to display the quintessential elements required for binding the dimetal cofactor. After thoroughly characterizing its structural, dynamic, spectroscopic, and functional properties, we added additional complexity in a rational stepwise manner to achieve increasingly sophisticated catalytic functions, ultimately demonstrating substrate-gated four-electron reduction of O2 to water. We also briefly describe the extension of these studies to the design of proteins that bind nonbiological metal cofactors (a synthetic porphyrin and a tetranuclear cluster), and a Zn2+/proton antiporting membrane protein. Together these studies demonstrate a successful and generally applicable strategy for de novo metalloprotein design, which might indeed mimic the process by which primordial metalloproteins evolved. We began the design process with a highly symmetrical backbone and binding site, by using point-group symmetry to assemble the secondary structures that position the amino acid side chains required for binding. The resulting models provided a rough starting point and initial parameters for the subsequent precise design of the final protein using modern methods of computational protein design. Unless the desired site is itself symmetrical, this process requires reduction of the symmetry or lifting it altogether. Nevertheless, the initial symmetrical structure can be helpful to restrain the search space during assembly of the backbone. Finally, the methods described here should be generally applicable to the design of highly stable and robust catalysts and sensors. There is considerable potential in combining the efficiency and knowledge base associated with homogeneous metal catalysis with the programmability, biocompatibility, and versatility of proteins. While the work reported here focuses on testing and learning the principles of natural metalloproteins by designing and studying proteins one at a time, there is also considerable potential for using designed proteins that incorporate both biological and nonbiological metal ion cofactors for the evolution of novel catalysts.

Dataset Information

Oxygen reactivity of the biferrous site in the de novo designed four helix bundle peptide DFsc: nature of the "intermediate" and reaction mechanism.

Publications

Oxygen reactivity of the biferrous site in the de novo designed four helix bundle peptide DFsc: nature of the "intermediate" and reaction mechanism.

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