Poster abstracts

Poster number 46 submitted by Effie Miller

Investigating molecular determinants of cofactor assembly and metal specificity in R2lox, a cambialistic Mn/Fe metalloprotein

Effie K. Miller (OSBP), Zachary R. Smith (Chemistry and Biochemistry, Ohio State University), Pearson T. Maugeri (Biophysics), Hannah Shafaat (OSBP)

Abstract:
As one of only two redox-active Mn/Fe proteins, the R2-like ligand-binding oxidase (R2lox) spontaneously and selectively forms a Mn/Fe cofactor in vitro, going against the commonly accepted Irving-Williams series for metal-binding affinities.1 In the presence of Fe alone, R2lox assembles a more canonical Fe/Fe cofactor.2 R2lox is capable of executing multi-electron chemistry, performing C-H bond oxidation upon O2 activation to generate an unprecedented tyrosine-valine crosslink within its scaffold,1,3 achieving this reaction along with a recently-discovered photodecarboxylation reaction with impressive control and selectivity.3 As such, R2lox presents a unique opportunity for investigating the molecular elements contributing to metal specificity, cofactor assembly, and chemical reactivity within a single protein scaffold.
In this work, we have utilized a multi-faceted approach including diverse biophysical and photochemical spectroscopies as well as molecular biology and protein engineering methods to address many of the questions surrounding the Mn/Fe protein systems. Initial examination of the WT Mn/Fe R2lox assembly mechanism using time-resolved spectroscopies, combined with global kinetic modeling, resulted in identification of two reaction intermediates with distinct kinetic profiles and optical and electronic signatures.4 To further examine metal binding and reactivity, point mutations in the primary and secondary metal coordination spheres have been introduced into the R2lox scaffold. Results from both WT R2lox and these variants shed light on the importance of the primary sphere hydrogen bonding network, and indicate different relative binding affinities for MnII and FeII. Taken together, this work provides characterization of the Mn/Fe R2lox assembly mechanism and reaction intermediates and sets the stage for using the R2lox scaffold to probe molecular determinants of heterobimetallic protein reactivity.

References:
(1) Andersson, C. S.; Högbom, M. Proc. Natl. Acad. Sci. 2009, 106 (14), 5633–5638.
(2) Griese, J. J.; Kositzki, R.; Schrapers, P.; Branca, R. M. M.; Nordström, A.; Lehtiö, J.; Haumann, M.; Högbom, M. J. Biol. Chem. 2015, 290 (42), 25254–25272.
(3) Maugeri, P. T.; Griese, J. J.; Branca, R. M.; Miller, E. K.; Smith, Z. R.; Eirich, J.; Högbom, M.; Shafaat, H. S. J. Am. Chem. Soc. 2018, 140 (4), 1471–1480.
(4) Miller, E. K.; Trivelas, N. E.; Maugeri, P. T.; Blaesi, E. J.; Shafaat, H. S. Biochemistry. 2017, 56 (26), 3369–3379.

Keywords: metalloprotein, spectroscopy, bioinorganic