for our reasoning. Regardless of this uncertainty, the efficiency of O 2 activation by the M248I variant significantly increased compared to wt 2NTDO by at least 40%. The comparison of 4-nitrotoluene turnover by wt 2NTDO and the M248I variant illustrates the drastic changes in efficiency of oxygenation caused by a single amino acid substitution. Ju and Parales showed that an additional substitution (L238V-M248I) evolved directly from the M248I variant. Indeed and as shown in Figure , the double-substituted variant L238V-M248I formed more oxygenation products per O 2 consumed (∆O 2 ) than both the wild-type and the singly-substituted