Bacteria readily oxidize alkanes utilizing the metalloenzyme alkane hydroxylase (AlkB) and thus the actions of
AlkB represent one of nature’s best defenses against the environmental effects of oil spills. Mammalian fatty
acids are also desaturated by integral membrane fatty acid desaturases that are structurally quite similar to AlkB.
It is not understood why AlkB catalyzes the addition of an OH group to an alkane while desaturases convert
single C-C bonds to double C=C bonds. AlkB and soluble methane monooxygenase (sMMO) both catalyze the
transformation of inert C-H bonds using a diiron catalyst but AlkB has a diiron catalyst with primarily nitrogen
atoms as ligands while sMMO has an oxygen-rich coordination site. The coordination environment is predicted
to influence the electronic structure and hence reactivity but the electronic structure of AlkB has not been
characterized. The long-term objective of this research effort is to understand how the structure of AlkB
determines the chemistry it affects and to use this knowledge to deepen our understanding of how biology
selectively activates molecular oxygen and catalyzes the oxidation of inert hydrocarbons. An associated
objective is to understand how structurally very similar biological motifs select between catalyzing the
hydroxylation of alkanes or their desaturation. The primary goal of the specific research proposed is obtain a
well-diffracting crystal of AlkB that will enable us to determine the three-dimensional structure of this enzyme.
Related goals are to spectroscopically characterize the ground state and reactive intermediates and to begin
explore AlkBs and related metalloenzymes from additional microorganisms. These goals enable us to test the
hypothesis that the structures of the active sites of all AlkBs and membrane-spanning desaturases are the same
but that structural differences in the substrate binding pocket control substrate selectivity and catalyst reactivity.
If successful, this work would have an impact on bioinorganic chemistry and environmental chemistry. A three-
dimensional structure of AlkB would help to answer many questions about the range of chemical motifs that
biology can use to oxidize alkanes. It would also answer questions about some of the chemical processes
utilized in the environmental response to oil spills.