Blue Type I Copper Centers vs Purple Cu A Centers 4439 5. Enzymes Employing a Combination of Different Types of Electron Transfer Centers 4439 5.1. Enzymes Using Both Heme and Cu as Electron Transfer Centers 4439 5.1.1. Cytochrome c and Cu A as Redox Partners to Cytochrome c Oxidases 4439 5.1.2. Cu A and Heme b as Redox Partners to Nitric Oxide Reductases 4440 5.1.3. Cytochrome c and Cu A as Redox Partners to Nitrous Oxide Reductases 4440 5.2. Enzymes Using Both Heme and Iron−Sulfur Clusters as Electron Transfer Centers 4440 5.2.1. As Redox Partners to the Cytochrome bc 1 Complex 4440 5.2.2. As Redox Partners to the Cytochrome b 6 f Complex 4442 5.2.3. As Redox Centers in Formate Dehydrogenases 4443 5.2.4. As Redox Centers in Nitrate Reductase 4443 6. Summary and Outlook 4443 Author Information 4445 Corresponding Author 4445 Author Contributions 4445 Notes 4445 Biographies 4445 Acknowledgments 4447 Abbreviations 4447 References 4447

Summary Rational design of functional enzymes with high turnovers is a significant challenge, especially those with complex active site and difficult reactions, such as in respiratory oxidases. Introducing 2 His and 1 Tyr into myoglobin resulted in designed enzymes that reduce O2 to H2O with > 1000 turnovers and minimal release of reactive oxygen species. This also showed that presence and positioning of Tyr, not Cu, are critical for activity.

Cytochrome c Oxidase (CcO) is known to catalyze the reduction of O2 to H2O efficiently with a much lower overpotential than most other O2 reduction catalysts. However, methods by which the enzyme fine-tunes the reduction potential (E°) of its active site and the corresponding influence on the O2 reduction activity are not well understood. In this work, we report systematic tuning of the heme E° in a functional model of CcO in myoglobin containing three histidines and one tyrosine in the distal pocket of heme. By removing hydrogen-bonding interactions between Ser92 and the proximal His ligand and a heme propionate, and increasing hydrophobicity of the heme pocket through Ser92Ala mutation, we have increased the heme E° from 95 ± 2 to 123 ± 3 mV. Additionally, replacing the native heme b in the CcO mimic with heme a analogs, diacetyl, monoformyl, and diformyl hemes, that posses electron-withdrawing groups, resulted in higher E° values of 175 ± 5, 210 ± 6, and 320 ± 10 mV, respectively. Furthermore, O2 consumption studies on these CcO mimics revealed a strong enhancement in O2 reduction rates with increasing heme E°. Such methods of tuning the heme E° through a combination of secondary sphere mutations and heme substitutions can be applied to tune E° of other heme proteins, allowing for comprehensive investigations of the relationship between E° and enzymatic activity.

Multielectron redox reactions often require multicofactor metalloenzymes to facilitate coupled electron and proton movement, but it is challenging to design artificial enzymes to catalyze these important reactions, owing to their structural and functional complexity. We report a designed heteronuclear heme-[4Fe-4S] cofactor in cytochrome peroxidase as a structural and functional model of the enzyme sulfite reductase. The initial model exhibits spectroscopic and ligand-binding properties of the native enzyme, and sulfite reduction activity was improved-through rational tuning of the secondary sphere interactions around the [4Fe-4S] and the substrate-binding sites-to be close to that of the native enzyme. By offering insight into the requirements for a demanding six-electron, seven-proton reaction that has so far eluded synthetic catalysts, this study provides strategies for designing highly functional multicofactor artificial enzymes.

Terminal oxidases catalyze four-electron reduction of oxygen to water, and the energy harvested is utilized to drive the synthesis of adenosine triphosphate. While much effort has been made to design a catalyst mimicking the function of terminal oxidases, most biomimetic catalysts have much lower activity than native oxidases. Herein we report a designed oxidase in myoglobin with an O2 reduction rate (52 s–1) comparable to that of a native cytochrome (cyt) cbb3 oxidase (50 s–1) under identical conditions. We achieved this goal by engineering more favorable electrostatic interactions between a functional oxidase model designed in sperm whale myoglobin and its native redox partner, cyt b5, resulting in a 400-fold electron transfer (ET) rate enhancement. Achieving high activity equivalent to that of native enzymes in a designed metalloenzyme offers deeper insight into the roles of tunable processes such as ET in oxidase activity and enzymatic function and may extend into applications such as more efficient oxygen reduction reaction catalysts for biofuel cells.

Metalloenzymes are among the major targets of protein design and engineering efforts aimed at attaining novel and efficient catalysis for biochemical transformation and biomedical applications, due to the diversity of functions imparted by the metallo-cofactors along with the versatility of the protein environment. Naturally evolved protein scaffolds can often serve as robust foundations for sustaining artificial active sites constructed by rational design, directed evolution, or a combination of the two strategies. Accumulated knowledge of structure-function relationship and advancement of tools such as computational algorithms and unnatural amino acids incorporation all contribute to the design of better metalloenzymes with catalytic properties approaching the needs of practical applications.

A major barrier to understanding the mechanism of nitric oxide reductases (NORs) is the lack of selective probe of NO binding to the non-heme FeB center. By replacing the heme in a biosynthetic model of NORs (L29H/F43H/V68E Mb), that structurally and functionally mimics NORs, with isostructural ZnPP, we report herein a study where the electronic structure and functional properties of the FeB-nitrosyl complex has been probed selectively. This approach allowed us to observe the first S=3/2 non-heme {FeNO}7 complex in a protein system. Such feats are not achievable in native NORs as these are complex membrane proteins containing multiple hemes. Detailed spectroscopic and computational studies show that the electronic state of the {FeNO}7 complex is best described as a HS ferrous iron (S=2) antiferromagnetically coupled to NO radical (S=1/2) [Fe2+-NO•]. The radical nature of the FeB-bound NO would facilitate N-N bond formation by radical coupling with the heme-bound NO. This finding, therefore, supports the proposed trans mechanism of NO reduction by NORs.

Metamorphic diamond in crustal rocks provides important information on the deep subduction of continental crust. Here, we present a new occurrence of diamond within the Seve Nappe Complex (SNC) of the Scandinavian Caledonides, on Åreskutan in Jämtland County, Sweden. Microdiamond is found in situ as single and composite (diamond+carbonate) inclusions within garnet, in kyanite‐bearing paragneisses. The rocks preserve the primary peak pressure assemblage of Ca,Mg‐rich garnet+phengite+kyanite+rutile, with polycrystalline quartz surrounded by radial cracks indicating breakdown of coesite. Calculated P–T conditions for this stage are 830–840 °C and 4.1–4.2 GPa, in the diamond stability field. The ultrahigh‐pressure (UHP) assemblage has been variably overprinted under granulite facies conditions of 850–860 °C and 1.0–1.1 GPa, leading to formation of Ca,Mg‐poor garnet+biotite+plagioclase+K‐feldspar+sillimanite+ilmenite+quartz. This overprint was the result of nearly isothermal decompression, which is corroborated by Ti‐in‐quartz thermometry. Chemical Th–U–Pb dating of monazite yields ages between 445 and 435 Ma, which are interpreted to record post‐UHP exhumation of the diamond‐bearing rocks. The new discovery of microdiamond on Åreskutan, together with other evidence of ultrahigh‐pressure metamorphism (UHPM) within gneisses, eclogites and peridotites elsewhere in the SNC, provide compelling arguments for regional (at least 200 km along strike of the unit) UHPM of substantial parts of this far‐travelled allochthon. The occurrence of UHPM in both rheologically weak (gneisses) and strong lithologies (eclogites, peridotites) speaks against the presence of large tectonic overpressure during metamorphism.