A Designed Functional Metalloenzyme that Reduces O2 to H2O with Over One Thousand Turnovers
Angew. Chem. Int. Ed. 2012, 51, 5589-5592, 10.1002/anie.201201981
Rational design of functional enzymes with a high number of turnovers is a challenge, especially those with a complex active site, such as respiratory oxidases. Introducing two His and one Tyr residues into myoglobin resulted in enzymes that reduce O2 to H2O with more than 1000 turnovers (red line, see scheme) and minimal release of reactive oxygen species. The positioning of the Tyr residue is critical for activity.
Max TON: 1056ee: ---Notes: Sperm whale myoglobin
Defining the Role of Tyrosine and Rational Tuning of Oxidase Activity by Genetic Incorporation of Unnatural Tyrosine Analogs
J. Am. Chem. Soc. 2015, 137, 4594-4597, 10.1021/ja5109936
While a conserved tyrosine (Tyr) is found in oxidases, the roles of phenol ring pKa and reduction potential in O2 reduction have not been defined despite many years of research on numerous oxidases and their models. These issues represent major challenges in our understanding of O2 reduction mechanism in bioenergetics. Through genetic incorporation of unnatural amino acid analogs of Tyr, with progressively decreasing pKa of the phenol ring and increasing reduction potential, in the active site of a functional model of oxidase in myoglobin, a linear dependence of both the O2 reduction activity and the fraction of H2O formation with the pKa of the phenol ring has been established. By using these unnatural amino acids as spectroscopic probe, we have provided conclusive evidence for the location of a Tyr radical generated during reaction with H2O2, by the distinctive hyperfine splitting patterns of the halogenated tyrosines and one of its deuterated derivatives incorporated at the 33 position of the protein. These results demonstrate for the first time that enhancing the proton donation ability of the Tyr enhances the oxidase activity, allowing the Tyr analogs to augment enzymatic activity beyond that of natural Tyr.
Ligand type: PorphyrinMax TON: 1200ee: ---Notes: Sperm whale myoglobin
Significant Increase of Oxidase Activity through the Genetic Incorporation of a Tyrosine–Histidine Cross-Link in a Myoglobin Model of Heme–Copper Oxidase
Angew. Chem. Int. Ed. 2012, 51, 4312-4316, 10.1002/anie.201108756
Top model: Heme–copper oxidase (HCO) contains a histidine–tyrosine cross‐link in its heme a3/CuB oxygen reduction center. A functional model of HCO was obtained through the genetic incorporation of the unnatural amino acid imiTyr, which mimics the Tyr–His cross‐link, and of the CuB site into myoglobin (see picture). Like HCO, this small soluble protein exhibits selective O2‐reduction activity while generating little reactive oxygen species.
Max TON: 1100ee: ---PDB: ---Notes: Sperm whale myoglobin
Systematic Tuning of Heme Redox Potentials and Its Effects on O2 Reduction Rates in a Designed Oxidase in Myoglobin
J. Am. Chem. Soc. 2014, 136, 11882-11885, 10.1021/ja5054863
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.
Max TON: 1600ee: ---Notes: Sperm whale myoglobin