Methylated Histidines Alter Tautomeric Preferences that Influence the Rates of Cu Nitrite Reductase Catalysis in Designed Peptides

Abstract

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 3methyl-histidine to create Cu nitrite reductases where δor 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 (k cat /K M) of native Cu nitrite reductase involve both substrate binding (K M) and catalysis (k cat).

Catalysis and Electron Transfer in De Novo Designed Helical Scaffolds

“…Adapted and reprinted from Refs. and with permission. Copyright 2018 Wiley‐VCH and 2019 American Chemical Society.…”

Katalyse und Elektronentransfer in helikalen De‐novo‐Gerüststrukturen

“…, und der primären koordinierenden Aminosäurereste (magenta) aus Lit. . Adaptiert mit Genehmigung aus Lit.…”

“…Adaptiert mit Genehmigung aus Lit. und . Copyright 2018 Wiley‐VCH und 2019 American Chemical Society.…”

Exploring the copper binding ability of Mets7 hCtr‐1 protein domain and His7 derivative: An insight in Michael addition catalysis

“…32,33 The same scaffolds were successfully employed in the preparation of cobalt-based hybrid electrocatalysts, for the selective six-electron reduction of nitrite and hydroxylamine intermediates to ammonium in aqueous buffer. 34,35 In our research group, we recently reported the catalytic ability of the complex between Mets7 and Cu(I) in Henry condensation reaction. 28 Mets7 is a methionine-rich motif naturally present in hCtr1copper transporters, devoted to the trafficking of cellular copper and recognized for being involved in inducing resistance to therapeutically approved platinum drugs by their sequestration.…”

“…Recently, starting from ATCUN motif, 30,31 a Cu (II)‐ and Ni (II)‐binding sequence found at the N ‐terminus of many naturally occurring proteins, ultrashort peptides were developed as iridium binding catalytic systems for asymmetric transfer hydrogenation of substituted ketones in water 32,33 . The same scaffolds were successfully employed in the preparation of cobalt‐based hybrid electrocatalysts, for the selective six‐electron reduction of nitrite and hydroxylamine intermediates to ammonium in aqueous buffer 34,35 …”