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Carotenoid cleavage dioxygenases (CCDs) are non-heme FeII enzymes that catalyze the oxidative cleavage of alkene bonds in carotenoids, stilbenoids, and related compounds. How these enzymes control the reaction of O2 with their alkene substrates is unclear. Here, we apply spectroscopy in conjunction with X-ray crystallography to define the iron coordination geometry of a model CCD, CAO1, in its resting state and following substrate binding and coordination sphere substitutions. Resting CAO1 exhibits a five-coordinate (5C), square pyramidal FeII center that undergoes steric distortion towards a trigonal bipyramidal geometry in the presence of piceatannol. Titrations with the O2-analog, nitric oxide (NO), show a >100-fold increase in iron-NO affinity upon substrate binding, defining a crucial role for the substrate in activating the FeII site for O2 reactivity. The importance of the 5C FeII structure for reactivity was probed through mutagenesis of the second-sphere Thr151 residue of CAO1, which occludes ligand binding at the sixth coordination position. A T151G substitution resulted in the conversion of the iron center to a six-coordinate (6C) state and a 135-fold reduction in apparent catalytic efficiency towards piceatannol compared to the wild-type enzyme. Substrate complexation resulted in partial 6C to 5C conversion, indicating solvent dissociation from the iron center. Additional substitutions at this site demonstrated a general functional importance of the occluding residue within the CCD superfamily. Taken together, these data suggest an ordered mechanism of CCD catalysis occurring via substrate-promoted solvent replacement by O2. CCDs thus represent a new class of mononuclear non-heme FeII enzymes. 

The primary and secondary coordination spheres of metal binding sites in metalloproteins have been investigated extensively, leading to the creation of high-performing functional metalloproteins; however, the impact of the overall structure of the protein scaffold on the unique properties of metalloproteins has rarely been studied. A primary example is the binuclear CuA center, an electron transfer cupredoxin domain of photosynthetic and respiratory complexes and, recently, a protein co-regulated with particulate methane and ammonia monooxygenases. The redox potential, Cu–Cu spectroscopic features, and a valence delocalized state of CuA are difficult to reproduce in synthetic models, and every artificial protein CuA center to-date has used a modified cupredoxin. Here we present a fully functional CuA center designed in a structurally non-homologous protein, cytochrome c peroxidase (CcP), by only two mutations (CuACcP). We demonstrate with UV-visible absorption, resonance Raman, and MCD spectroscopy that CuACcP is valence delocalized. CW and pulsed (HYSCORE) X-band EPR show it has a highly compact gz area and small Az hyperfine principal value with g and A tensors that resemble axially perturbed CuA. Stopped-flow kinetics found that CuA formation proceeds through a single T2Cu intermediate. The reduction potential of CuACcP is comparable to native CuA and can transfer electrons to a physiological redox partner. We built a structural model of the designed Cu binding site from EXAFS and validated it by mutation of coordinating Cys and His residues, revealing that a triad of residues (R48C, W51C, and His52) rigidly arranged on one α-helix is responsible for chelating the first Cu atom and that His175 stabilizes the binuclear complex by rearrangement of the CcP heme-coordinating helix. This design is a demonstration that a highly conserved protein fold is not uniquely necessary to induce certain characteristic physical and chemical properties to a metal redox center. 

The recent research developments on the active sites in Fe-zeolites for redox catalysis are discussed. Building on the characterisation of the α-Fe/α-O active sites in the beta and chabazite zeolites, we demonstrate a bottom-up approach to successfully understand and develop Fe-zeolite catalysts. We use the room temperature benzene to phenol reaction as a relevant example. We then suggest how the spectroscopic identification of other monomeric and dimeric iron sites could be tackled. The challenges in the characterisation of active sites and intermediates in NO X selective catalytic reduction catalysts and further development of catalysts for mild partial methane oxidation are briefly discussed.