A De Novo Designed Trimeric Metalloprotein as a Nip Model of the Acetyl-CoA Synthase.

We present a Nip site model of acetyl coenzyme-A synthase (ACS) within a de novo-designed trimer peptide that self-assembles to produce a homoleptic Ni(Cys)3 binding motif. Spectroscopic and kinetic studies of ligand binding demonstrate that Ni binding stabilizes the peptide assembly and produces a terminal NiI-CO complex. When the CO-bound state is reacted with a methyl donor, a new species is quickly produced with new spectral features. While the metal-bound CO is albeit unactivated, the presence of the methyl donor produces an activated metal-CO complex. Selective outer sphere steric modifications demonstrate that the physical properties of the ligand-bound states are altered differently depending on the location of the steric modification above or below the Ni site.

A De Novo-Designed Artificial Metallopeptide Hydrogenase: Insights into Photochemical Processes and the Role of Protonated Cys.

Hydrogenase enzymes produce H2 gas, which can be a potential source of alternative energy. Inspired by the [NiFe] hydrogenases, we report the construction of a de novo-designed artificial hydrogenase (ArH). The ArH is a dimeric coiled coil where two cysteine (Cys) residues are introduced at tandem a/d positions of a heptad to create a tetrathiolato Ni binding site. Spectroscopic studies show that Ni binding significantly stabilizes the peptide producing electronic transitions characteristic of Ni-thiolate proteins. The ArH produces H2 photocatalytically, demonstrating a bell-shaped pH-dependence on activity. Fluorescence lifetimes and transient absorption spectroscopic studies are undertaken to elucidate the nature of pH-dependence, and to monitor the reaction kinetics of the photochemical processes. pH titrations are employed to determine the role of protonated Cys on reactivity. Through combining these results, a fine balance is found between solution acidity and the electron transfer steps. This balance is critical to maximize the production of NiI -peptide and protonation of the NiII -H- intermediate (Ni-R) by a Cys (pKa ≈6.4) to produce H2 .

Biosynthetic Approaches towards the Design of Artificial Hydrogen-Evolution Catalysts.

Hydrogen is a clean and sustainable form of fuel that can minimize our heavy dependence on fossil fuels as the primary energy source. The need of finding greener ways to generate H2 gas has ignited interest in the research community to synthesize catalysts that can produce H2 by the reduction of H+ . The natural H2 producing enzymes hydrogenases have served as an inspiration to produce catalytic metal centers akin to these native enzymes. In this article we describe recent advances in the design of a unique class of artificial hydrogen evolving catalysts that combine the features of the active site metal(s) surrounded by a polypeptide component. The examples of these biosynthetic catalysts discussed here include i) assemblies of synthetic cofactors with native proteins; ii) peptide-appended synthetic complexes; iii) substitution of native cofactors with non-native cofactors; iv) metal substitution from rubredoxin; and v) a reengineered Cu storage protein into a Ni binding protein. Aspects of key design considerations in the construction of these artificial biocatalysts and insights gained into their chemical reactivity are discussed.

The oxygen reactivity of an artificial hydrogenase designed in a reengineered copper storage protein.

The O2 reactivity of an artificial biomolecular hydrogenase, the nickel binding protein (NBP) is investigated. Kinetic analyses revealed a complete 4e- reduction of O2 to H2O under catalytic conditions with associated k0 for ET in the order of 10-6 cm s-1. Protein destabilization and S oxygenation are contributing factors to the deactivation of NBP under oxic conditions. Computational studies provided insight into the S oxygenation and the reaction intermediates of a proposed mechanistic pathway for O2 activation by NBP.

Redesign of a Copper Storage Protein into an Artificial Hydrogenase.

We report the construction of an artificial hydrogenase (ArH) by reengineering a Cu storage protein (Cspl) into a Ni-binding protein (NBP) employing rational metalloprotein design. The hypothesis driven design approach involved deleting existing Cu sites of Csp1 and identification of a target tetrathiolate Ni binding site within the protein scaffold followed by repacking the hydrophobic core. Guided by modeling, the NBP was expressed and purified in high purity. NBP is a well-folded and stable construct displaying native-like unfolding behavior. Spectroscopic and computational studies indicated that the NBP bound nickel in a distorted square planar geometry that validated the design. Ni(II)-NBP is active for photo-induced H2 evolution following a reductive quenching mechanism. Ni(II)-NBP catalyzed H+ reduction to H2 gas electrochemically as well. Analysis of the catalytic voltammograms established a proton-coupled electron transfer (PCET) mechanism. Electrolysis studies confirmed H2 evolution with quantitative Faradaic yields. Our studies demonstrate an important scope of rational metalloprotein design that allows imparting functions into protein scaffolds that have natively not evolved to possess the same function of the target metalloprotein constructs.

Intrinsically fluorescent gold nanoclusters stabilized within a copper storage protein that follow the Irving-Williams trend in metal ion sensing.

Creating new environmentally friendly and non-toxic biomaterials with novel properties is required for numerous applications in healthcare and sensing. Protein bound gold nanoclusters constitute one such class of materials that offer promise in fluorescence imaging and sensing applications. However, unlike alkane thiol-protected gold nanoclusters, the number of protein-templated gold nanoclusters with such properties is limited and there is a need to expand the repertoire of such attractive hybrid quantum clusters. Herein, we report the synthesis, characterization, and applications of new fluorescent gold nanoclusters with tunable emission properties including blue, orange, and red, within a four-helix bundle copper storage protein (Csp1). The template protein consists of 13 cysteines along the length of the helix, which are suitable ligands to template Au and stabilize the resulting 14-19 atom clusters within the protein. The resulting clusters were extensively characterized by employing spectroscopic, microscopic and other analytical methods. The optical emission, relative quantum yields, and the excited state lifetime of the clusters are shown to depend on synthetic conditions. The clusters were found to be sensitive to the ppm level of transition metal ions with the quenching capabilities following the Irving-Williams series of metals (Co2+ < Ni2+ < Cu2+), which is rationalized based on the relative affinities of transition metals for a given set of ligands. The clusters were also found to be stable across the pH range 4-8.5 which, along with tunable emission properties paves the path for live bio-imaging and bio-sensing applications under physiological conditions.