posted on 2020-03-09, 14:41authored byEffie
C. Kisgeropoulos, Julia J. Griese, Zachary R. Smith, Rui M. M. Branca, Camille R. Schneider, Martin Högbom, Hannah S. Shafaat
Heterobimetallic Mn/Fe proteins
represent a new cofactor
paradigm in bioinorganic chemistry and pose countless outstanding
questions. The assembly of the active site defies common chemical
convention by contradicting the Irving–Williams series, while
the scope of reactivity remains unexplored. In this work, the assembly
and C–H bond activation process in the Mn/Fe R2-like ligand-binding
oxidase (R2lox) protein is investigated using a suite of biophysical
techniques, including time-resolved optical spectroscopy, global kinetic
modeling, X-ray crystallography, electron paramagnetic resonance spectroscopy,
protein electrochemistry, and mass spectrometry. Selective metal binding
is found to be under thermodynamic control, with the binding sites
within the apo-protein exhibiting greater MnII affinity
than FeII affinity. The comprehensive analysis of structure
and reactivity of wild-type R2lox and targeted primary and secondary
sphere mutants indicate that the efficiency of C–H bond activation
directly correlates with the Mn/Fe cofactor reduction potentials and
is inversely related to divalent metal binding affinity. These findings
suggest the R2lox active site is precisely tuned for achieving both
selective heterobimetallic binding and high levels of reactivity
and offer a mechanism to examine the means by which proteins achieve
appropriate metal incorporation.