Project Summary
The oxidation of carbon-hydrogen (C-H) bonds is a central biochemical process required by all aerobic
organisms. The microbial conversion of these bonds endows potent bioactivity on natural products and enable
pathogens to thrive on otherwise inert host-derived biomolecules. These reactions are currently known to be
accomplished by a short list of cofactors that include heme, nonheme iron, and copper. While manganese
cofactors perform difficult oxidation reactions, including water oxidation within Photosystem II, they are generally
not known to be used for C-H bond activation. We recently discovered that the 2-aminoisobutyric acid
hydroxylase from Rhodococcus wratislaviensis, AibH1H2, requires manganese to functionalize a strong,
aliphatic C-H bond (BDE = 100 kcal/mol). Structural and spectroscopic studies of this enzyme revealed a redox-
active, heterobimetallic manganese-iron active site at the locus of O2 activation and substrate coordination. This
result expands the known reactivity of biological manganese-iron cofactors, which was previously restricted to
single electron transfer or stoichiometric protein oxidation. Since the AibH1H2 cofactor is supported by a protein
fold distinct from typical, well-studied bimetallic oxygenases, our proposed research centers on the
characterization of ill-defined members of this protein family. Our preliminary results indicate that many of these
proteins harbor two active-site metal ions, behave as monooxygenases and, in some cases, display enhanced
enzymatic activity when manganese ions are preferentially incorporated within the active site. The core scientific
hypothesis for this study is that the unique sequence and structural features of this emergent class of
monooxygenases endows embedded manganese ions with otherwise unknown biological reactivity. To
investigate this hypothesis, the specific aims of this study are to (1) understand the signature amino acid motifs
that differentiate these monooxygenases from their ancestral, structurally-related amidohydrolases, (2)
characterize the geometric and electronic structures of mixed iron/manganese-containing enzymatic
intermediates, and (3) evaluate the thermodynamic and kinetic competence of cambialistic Mn/Fe
monooxygenases. The resulting enzymatic information, methods, and structures will be of specific interest to
those in the fields of metallo-enzymology, structural biology, and synthetic bioinorganic chemistry, and of broad
interest to microbiologists in general.