Poster Presentation 21st International Conference on Biological Inorganic Chemistry 2025

Histidine Deprotonation and Copper(III) at Coupled Binuclear Copper Sites: The Potential Role of N-H Protons (#540)

Tao A. G. Large 1 , T. Daniel P. Stack 1
  1. Department of Chemistry, Stanford University, Palo Alto, CA, United States

Multinuclear copper enzymes such as tyrosinases, catechol oxidases, and multicopper oxidases reduce dioxygen exclusively at histidine-ligated coupled binuclear copper sites, which are conserved across aerobic life.1 While the first two-electron reduction of dioxygen is well-established (2Cu1+ + dioxygen → 2Cu2+ + peroxide), models for the second two-electron step are evolving rapidly; converging consensus suggests this step may present a first example of a biological system able to access copper(III).2-6 This talk will present synthetic precedent for a viable copper(III) intermediate that forms when deprotonation of ligating histidine can occur.2

 

The implications of histidine deprotonation can be challenging to probe directly in proteins but can be tested directly using appropriate synthetic systems. The effects are not subtle. We find that histidine deprotonation reduces dioxygen from peroxide to oxides, and oxidizes both copper centers to copper(III). This creates an electrophilic LUMO positioned appropriately to accept substrate electrons, and a deprotonated histidine positioned appropriately to act as a substrate-deprotonating base.2

 

A capacity for copper centers to switch from one-electron to two-electron shuttles through histidine hints at a provocative but simple alternative mechanism for tyrosinases, which finds theoretical support. Electrophilic aromatic substitution through a formal copper(III) intermediate accessed by even transient histidine deprotonation leads to only reasonable barriers, with deprotonation contributing to catalysis by up to 15 kcal•mol-1.

 

Given the conserved structures of binuclear copper sites, we suggest that copper(III) accessibility though histidine deprotonation may be more viable than generally appreciated.

 

  1. Solomon, E. I.; Heppner, D. E.; Johnston, E. M.; Ginsbach, J. W.; Cirera, J.; Qayyum, M.; Kieber-Emmons, M. T.; Kjaergaard, C. H.; Hadt, R. G.; Tian, L. Copper Active Sites in Biology. Chemical Reviews 2014, 114 (7), 3659-3853.
  2. Large, T. A. G.; Keown, W.; Gary, J. B.; Chiang, L.; Stack, T. D. P. Imidazolate-Stabilized Cu(III): Dioxygen to Oxides at Type 3 Copper Sites. Angewandte Chemie International Edition 2025, 64 (5), e202416967.
  3. Kipouros, I.; Stańczak, A.; Dunietz, E. M.; Ginsbach, J. W.; Srnec, M.; Rulíšek, L.; Solomon, E. I. Experimental Evidence and Mechanistic Description of the Phenolic H-Transfer to the Cu2O2 Active Site of oxy-Tyrosinase. Journal of the American Chemical Society 2023, 145 (42), 22866-22870.
  4. Kipouros, I.; Solomon, E. I. New mechanistic insights into coupled binuclear copper monooxygenases from the recent elucidation of the ternary intermediate of tyrosinase. FEBS Letters 2023, 597 (1), 65-78.
  5. Kipouros, I.; Stańczak, A.; Ginsbach, J. W.; Andrikopoulos, P. C.; Rulíšek, L.; Solomon, E. I. Elucidation of the tyrosinase/O2/monophenol ternary intermediate that dictates the monooxygenation mechanism in melanin biosynthesis. Proceedings of the National Academy of Sciences 2022, 119 (33), e2205619119.
  6. Stańczak, A.; Kipouros, I.; Eminger, P.; Dunietz, E. M.; Solomon, E. I.; Rulíšek, L. Coupled binuclear copper sites in biology: An experimentally-calibrated computational perspective. Coordination Chemistry Reviews 2025, 525, 216301.