The bottom-up design and construction of functional metalloproteins remains a formidable task in biomolecular design. While numerous strategies have been used to create new metalloproteins, preexisting knowledge of the tertiary and quaternary protein structure is often required to generate suitable platforms for robust metal coordination and activity. Here we report an alternative and easily implemented approach (Metal Active Sites by Covalent Tethering or MASCoT) whereby folded protein building blocks are linked by a single disulfide bond to create diverse metal coordination environments within evolutionarily naïve protein-protein interfaces. Metalloproteins generated with this strategy uniformly bind a wide array of first-row transition metal ions (Mn II , Fe II , Co II , Ni II , Cu II , Zn II and vanadyl) with physiologically relevant thermodynamic affinities (dissociation constants ranging from 700 nM for Mn II to 50 fM for Cu II ). MASCoT readily affords coordinatively unsaturated metal centers, including a five-His coordinated non-heme Fe site, and well-defined binding pockets that can accommodate modifications and enable coordination of exogenous ligands like nitric oxide to the interfacial metal center.

Non-heme iron oxygenases utilize dioxygen to accomplish challenging chemical oxidations. Further understanding of the Fe-O2 intermediates implicated in these processes is challenged by their highly transient nature. To that end, we have developed a ligand platform featuring phosphinimide donors intended to stabilize oxidized, high-spin iron complexes. O2 exposure of single crystals of a threecoordinate Fe(II) complex of this framework allowed for in crystallo trapping of a terminally-bound Fe-O2 complex suitable for XRD characterization. Spectroscopic and computational studies of this species support a high-spin Fe(III) center antiferromagnetically coupled to a superoxide ligand, similar to that proposed for numerous non-heme iron oxygenases. In addition to the stability of this synthetic Fe-O2 complex, its ability to engage in a range of stoichiometric and catalytic oxidation processes demonstrates that this iron-phosphinimide system is primed for development in modelling oxidizing bioinorganic intermediates and green oxidation chemistry.

Electrons added to TiO 2 and other semiconductors often occupy trap states, whose reactivity can determine the catalytic and stoichiometric chemistry of the material. We previously showed that reduced aqueous colloidal TiO 2 nanoparticles have two distinct classes of thermally equilibrated trapped electrons, termed Red/e − and Blue/e − . Presented here are parallel optical and electron paramagnetic resonance (EPR) kinetic studies of the reactivity of these electrons with solution-based oxidants. Optical stopped-flow measurements monitoring the reactions of TiO 2 /e − with substoichiometric oxidants showed a surprising pattern: an initial fast (seconds) decrease in TiO 2 /e − absorbance followed by a secondary, slow (minutes) increase in the broad TiO 2 /e − optical feature. The analysis revealed that the fast decrease is due to the preferential oxidation of the Red/e − trap states and the slow increase results from the re-equilibration of electrons from Blue/e − to Red/e − states. This kinetic model was confirmed by freeze-quench EPR measurements. Quantitative analysis of the kinetic data demonstrated that Red/e − react ∼5 times faster than Blue/e − with the nitroxyl radical oxidant 4-methoxy-2,2,6,6-tetramethyl-1-piperidinyloxyl (4-MeO-TEMPO). Similar reactivity patterns were also observed in oxidations of TiO 2 /e − by O 2 , which like 4-MeO-TEMPO is a proton-coupled electron transfer (PCET) oxidant, and by the pure electron transfer (ET) oxidant potassium triiodide (KI 3 ). This suggests that the faster intrinsic reactivity of one trap state over another on the seconds− minutes time scale is likely a general feature of reduced TiO 2 reactivity. This differential trap-state reactivity is likely to influence the performance of TiO 2 in photochemical/electrochemical devices, and it suggests an opportunity for tuning catalysis.

Lignolytic peroxidases use an electron transfer (ET) pathway that involves amino acid-mediated substrate oxidation at the surface of the protein rather than at an embedded heme site. In many of these peroxidases, redox catalysis takes place at a substrate accessible tyrosine or tryptophan (Trp) amino acid. Here, we describe new mutants of cytochrome c peroxidase (CcP) that were designed to incorporate a Trp-based "wire" that can move oxidizing equivalents from the heme to the protein surface. Three mutant CcP proteins were expressed and characterized: A193W, Y229W, and A193W/Y229W. These mutants can oxidize veratryl alcohol substrate with turnover numbers greater than wild type CcP using HO as an oxidant. The A193W/Y229W mutant is the most active. However, the reactivity is still less than typical lignin peroxidases at pH 8. The redox reactivity of these proteins is analysed using semiclassical electron transfer theory. An electron hopping mechanism is possible for A193W/Y229W mutant. These data suggest that artificial chains of aromatic amino acids can support hole transfer from embedded sites to protein surfaces for catalytic redox reactions.