Rational design of a hexameric protein assembly stabilized by metal chelation.

Protein-based self-assembled nanostructures hold tremendous promise as smart materials. One strategy to control the assembly of individual protein modules takes advantage of the directionality and high affinity bonding afforded by metal chelation. Here, we describe the use of 2,2'-bipyridine units (Bpy) as side chains to template the assembly of large structures (MW approx. 35 000 Da) in a metal-dependent manner. The structures are trimers of independently folded 3-helix bundles, and are held together by 2 Me(Bpy)3 complexes. The assemblies are stable to thermal denaturation, and are more than 90% helical at 90°C. Circular dichroism spectroscopy shows that one of the 2 possible (Bpy)3 enantiomers is favored over the other. Because of the sequence pliability of the starting peptides, these constructs could find use to organize functional groups at controlled positions within a supramolecular assembly.

Reengineering cyt b562 for hydrogen production: A facile route to artificial hydrogenases.

Bioinspired, protein-based molecular catalysts utilizing base metals at the active are emerging as a promising avenue to sustainable hydrogen production. The protein matrix modulates the intrinsic reactivity of organometallic active sites by tuning second-sphere and long-range interactions. Here, we show that swapping Co-Protoporphyrin IX for Fe-Protoporphyrin IX in cytochrome b562 results in an efficient catalyst for photoinduced proton reduction to molecular hydrogen. Further, the activity of wild type Co-cyt b562 can be modulated by a factor of 2.5 by exchanging the coordinating methionine with alanine or aspartic acid. The observed turnover numbers (TON) range between 125 and 305, and correlate well with the redox potential of the Co-cyt b562 mutants. The photosensitized system catalyzes proton reduction with high efficiency even under an aerobic atmosphere, implicating its use for biotechnological applications. This article is part of a Special Issue entitled Biodesign for Bioenergetics--the design and engineering of electronic transfer cofactors, proteins and protein networks, edited by Ronald L. Koder and J.L. Ross Anderson.

Design of Redox-Active Peptides: Towards Functional Materials.

In nature, the majority of processes that occur in the cell involve the cycling of electrons and protons, changing the reduction and oxidation state of substrates to alter their chemical reactivity and usefulness in vivo. One of the most relevant examples of these processes is the electron transport chain, a series of oxidoreductase proteins that shuttle electrons through well-defined pathways, concurrently moving protons across the cell membrane. Inspired by these processes, researchers have sought to develop materials to mimic natural systems for a number of applications, including fuel production. The most common cofactors found in proteins to carry out electron transfer are iron sulfur clusters and porphyrin-like molecules. Both types have been studied within natural proteins, such as in photosynthetic machinery or soluble electron carriers; in parallel, an extensive literature has developed over recent years attempting to model and study these cofactors within peptide-based materials. This chapter will focus on major designs that have significantly advanced the field.

Modulation of cluster incorporation specificity in a de novo iron-sulfur cluster binding peptide.

iron-sulfur cluster binding proteins perform an astounding variety of functions, and represent one of the most abundant classes of metalloproteins. Most often, they constitute pairs or chains and act as electron transfer modules either within complex redox enzymes or within small diffusible proteins. We have previously described the design of a three-helix bundle that can bind two clusters within its hydrophobic core. Here, we use single-point mutations to exchange one of the Cys ligands coordinating the cluster to either Leu or Ser. We show that the mutants modulate the redox potential of the clusters and stabilize the [3Fe-4S] form over the [4Fe-4S] form, supporting the use of model iron-sulfur cluster proteins as modules in the design of complex redox enzymes.

A de novo designed 2[4Fe-4S] ferredoxin mimic mediates electron transfer.

[Fe-S] clusters, nature's modular electron transfer units, are often arranged in chains that support long-range electron transfer. Despite considerable interest, the design of biomimetic artificial systems emulating multicluster-binding proteins, with the final goal of integrating them in man-made oxidoreductases, remains elusive. Here, we report a novel bis-[4Fe-4S] cluster binding protein, DSD-Fdm, in which the two clusters are positioned within a distance of 12 Å, compatible with the electronic coupling necessary for efficient electron transfer. The design exploits the structural repeat of coiled coils as well as the symmetry of the starting scaffold, a homodimeric helical protein (DSD). In total, eight hydrophobic residues in the core of DSD were replaced by eight cysteine residues that serve as ligands to the [4Fe-4S] clusters. Incorporation of two [4Fe-4S] clusters proceeds with high yield. The two [4Fe-4S] clusters are located in the hydrophobic core of the helical bundle as characterized by various biophysical techniques. The secondary structure of the apo and holo proteins is conserved; further, the incorporation of clusters results in stabilization of the protein with respect to chemical denaturation. Most importantly, this de novo designed protein can mimic the function of natural ferredoxins: we show here that reduced DSD-Fdm transfers electrons to cytochrome c, thus generating the reduced cyt c stoichiometrically.

De novo design of functional proteins: Toward artificial hydrogenases.

Over the last 25 years, de novo design has proven to be a valid approach to generate novel, well-folded proteins, and most recently, functional proteins. In response to societal needs, this approach is been used increasingly to design functional proteins developed with an eye toward sustainable fuel production. This review surveys recent examples of bioinspired de novo designed peptide based catalysts, focusing in particular on artificial hydrogenases.

Cobalt substitution supports an inner-sphere electron transfer mechanism for oxygen reduction in pea seedling amine oxidase.

Copper amine oxidases (CAOs) are a large family of proteins that use molecular oxygen to oxidize amines to aldehydes with the concomitant production of hydrogen peroxide and ammonia. CAOs utilize two cofactors for this reaction: topaquinone (TPQ) and a Cu(II) ion. Two mechanisms for oxygen reduction have been proposed for these enzymes. In one mechanism (involving inner-sphere electron transfer to O(2)), Cu(II) is reduced by TPQ, forming Cu(I), to which O(2) binds, forming a copper-superoxide complex. In an alternative mechanism (involving outer-sphere electron transfer to O(2)), O(2) is directly reduced by TPQ, without reduction of Cu(II). Substitution of Cu(II) with Co(II) has been used to distinguish between the two mechanisms in several CAOs. Because it is unlikely that Co(II) could be reduced to Co(I) in this environment, an inner-sphere mechanism, as described above, is prevented. We adapted metal replacement methods used for other CAOs to the amine oxidase from pea seedlings (PSAO). Cobalt-substituted PSAO (CoPSAO) displayed nominal catalytic activity: k(cat) is 4.7% of the native k(cat), and K(M) (O(2)) for CoPSAO is substantially (22-fold) higher. The greatly reduced turnover number for CoPSAO suggests that PSAO uses the inner-sphere mechanism, as has been predicted from (18)O isotope effect studies (Mukherjee et al. in J Am Chem Soc 130:9459-9473, 2008), and is catalytically compromised when constrained to operate via outer-sphere electron transfer to O(2). This study, together with previous work, provides strong evidence that CAOs use both proposed mechanisms, but each homolog may prefer one mechanism over the other.

Light-Driven CO2 Reduction by Co-Cytochrome b 562.

The current trend in atmospheric carbon dioxide concentrations is causing increasing concerns for its environmental impacts, and spurring the developments of sustainable methods to reduce CO2 to usable molecules. We report the light-driven CO2 reduction in water in mild conditions by artificial protein catalysts based on cytochrome b 562 and incorporating cobalt protoporphyrin IX as cofactor. Incorporation into the protein scaffolds enhances the intrinsic reactivity of the cobalt porphyrin toward proton reduction and CO generation. Mutations around the binding site modulate the activity of the enzyme, pointing to the possibility of further improving catalytic activity through rational design or directed evolution.

Repeat proteins as versatile scaffolds for arrays of redox-active FeS clusters.

Arrays of one, two and four electron-transfer active [4Fe-4S] clusters were constructed on modular tetratricopeptide repeat protein scaffolds, with the number of clusters determined solely by the size of the scaffold. The constructs show reversible redox activity and transient charge stabilization necessary to facilitate charge transfer.

Protein secondary-shell interactions enhance the photoinduced hydrogen production of cobalt protoporphyrin IX.

Hydrogen is an attractive fuel with potential for production scalability, provided that inexpensive, efficient molecular catalysts utilizing base metals can be developed for hydrogen production. Here we show for the first time that cobalt myoglobin (CoMyo) catalyzes hydrogen production in mild aerobic conditions with turnover number of 520 over 8 hours. Compared to free Co-protoporphyrin IX, incorporation into the myoglobin scaffold results in a 4-fold increase in photoinduced hydrogen production activity. Engineered variants in which specific histidine resides in proximity of the active site were mutated to alanine result in modulation of the catalytic activity, with the H64A/H97A mutant displaying activity 2.5-fold higher than wild type. Our results demonstrate that protein scaffolds can augment and modulate the intrinsic catalytic activity of molecular hydrogen production catalysts.