Unexpected Roles of a Tether Harboring a Tyrosine Gatekeeper Residue in Modular Nitrite Reductase Catalysis

Tobias Hedison, Rajesh Shenoy, Andreea Iulia Iorgu, Derren Heyes, Karl Fisher, Gareth Wright, Sam Hay, Robert Roy Eady, Svetlana Antonyuk, S. Samar Hasnain, Nigel S. Scrutton

Research output: Contribution to journalArticlepeer-review


It is generally assumed that tethering enhances rates of electron harvesting and delivery to active sites in multi-domain enzymes by proximity and sampling mechanisms. Here, we explore this idea in a tethered 3-domain, trimeric copper-containing nitrite reductase. By reverse engineering, we find that tethering does not enhance the rate of electron delivery from its pendant cytochrome c to the catalytic copper-containing core. Using a linker that harbors a gatekeeper tyrosine in a nitrite access channel, the tethered haem domain enables catalysis by other mechanisms. Tethering communicates the redox state of the haem to the distant T2Cu center that helps initiate substrate binding for catalysis. It also tunes copper reduction potentials, suppresses reductive enzyme inactivation, enhances enzyme affinity for substrate and promotes inter-copper electron transfer. Tethering has multiple unanticipated beneficial roles, the combination of which fine-tunes function beyond simplistic mechanisms expected from proximity and restrictive sampling models.
Original languageEnglish
JournalACS Catalysis
Early online date29 May 2019
Publication statusPublished - 2019


  • Copper nitrite reductase
  • tethering
  • enzyme catalysis
  • electron transfer
  • modular enzyme architecture
  • intraprotein electron transfer
  • interprotein electron transfer
  • protein dynamics

Research Beacons, Institutes and Platforms

  • Manchester Institute of Biotechnology


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