Free amino and carboxylic groups of the horse heart cytochrome C were modified by chemical reaction with methyl, trimethylsilyl (TMS), and poly(ethylene)glycol (PEG) moieties. As a consequence of chemical modification, the heme environment (active site) was altered. The kinetic constants and substrate specificities were determined for the differently modified cytochromes. A cytochrome with a double modification (PEG on free amino groups and methyl esters on carboxylic groups) was able to oxidize 17 aromatic compounds from 20 tested while the unmodified protein was only able to oxidize 8 compounds. This work shows that chemical modification of a biocatalyst could be a tool for the design of a new biocatalyst with environmental purposes.