Bacterial alkane hydroxylases have tremendous potential as biocatalysts for stereo- and regio-selective transformation of unreactive alkanes and for bioremediation, as they allow some bacteria to grow in oil-contaminated environments. This work will determine the molecular basis for catalysis and specificity of non-heme diiron medium-chain alkane hydroxylase by solving its first crystal structure and by carrying out interlocking biochemical experiments with chemical probes. Identification of the active site residues that contact the substrate will pave the way to rational design of this enzyme with the aim to increase its biocatalytic potential for modification of industrially important chemical precursors and biodegradation of spilt oils.