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Methylated Histidines Alter Tautomeric Preferences that Influence the Rates of Cu Nitrite Reductase Catalysis in Designed Peptides
- Koebke, Karl J., Yu, Fangting, Van Stappen, Casey, Pinter, Tyler B. J., Deb, Aniruddha, Penner-Hahn, James E., Pecoraro, Vincent L.
- Journal of the American Chemical Society 2019 v.141 no.19 pp. 7765-7775
- catalysts, catalytic activity, copper, crystal structure, electron transfer, enzyme kinetics, histidine, isomers, methylation, mutation, nitrite reductase, nitrites, nitrogen, peptides, proteins
- Copper proteins have the capacity to serve as both redox active catalysts and purely electron transfer centers. A longstanding question in this field is how the function of histidine ligated Cu centers are modulated by δ vs ε-nitrogen ligation of the imidazole. Evaluating the impact of these coordination modes on structure and function by comparative analysis of deposited crystal structures is confounded by factors such as differing protein folds and disparate secondary coordination spheres that make direct comparison of these isomers difficult. Here, we present a series of de novo designed proteins using the noncanonical amino acids 1-methyl-histidine and 3-methyl-histidine to create Cu nitrite reductases where δ- or ε-nitrogen ligation is enforced by the opposite nitrogen’s methylation as a means of directly comparing these two ligation states in the same protein fold. We find that ε-nitrogen ligation allows for a better nitrite reduction catalyst, displaying 2 orders of magnitude higher activity than the δ-nitrogen ligated construct. Methylation of the δ nitrogen, combined with a secondary sphere mutation we have previously published, has produced a new record for efficiency within a homogeneous aqueous system, improving by 1 order of magnitude the previously published most efficient construct. Furthermore, we have measured Michaelis–Menten kinetics on these highly active constructs, revealing that the remaining barriers to matching the catalytic efficiency (kcₐₜ/KM) of native Cu nitrite reductase involve both substrate binding (KM) and catalysis (kcₐₜ).