Indian Journal of Chemistry Vol. 50A, March-April 2011, pp. 438-446 Interaction of gammaxene with site specific mutants of cytochrome P450 cam Saptaswa Sen, Soumen Kanti Manna & Shyamalava Mazumdar* Department of Chemical Sciences, Tata Institute of Fundamental Research, Homi Bhabha Road, Colaba, Mumbai 400 005, India Email: shyamal@tifr.res.in Received 1 November 2010; accepted 18 December 2010 Cytochrome P450 cam (CYP101) from soil bacteria Pseudomonas putida, is one of the most well studied heme-b monoxygenase. A large number of X-ray crystal structures of this enzyme and its mutants are now available with different types of substrates that can be used to study the topology of the active site of the enzyme. We have a continuing interest in applying the current knowledge of cytochrome P450 cam -substrate recognition to rationally design the enzyme for the biotransformation of unnatural substrates, like gammaxene, with the long-term aim of applications in bioremediation of environmental contaminants. Comparison of the structure of target substrate with that of camphor, the natural substrate, led us to engineer the heme active-site and we have found that binding affinities significantly are increased for Y96F, Y96F/L244A and Y96F/T101V mutants. This has shown the way to new functions of the enzymes, which not only has provided a novel approach to the study of the mechanism of this complex super-family of enzymes, but has also led to the discovery of green biocatalysts for environmental applications. Keywords: Bioinorganic chemistry, Gammaxene, Cytochrome P450 cam , Heme, Oxygenase, Site specific mutagenesis Polyhalogenated hydrocarbons are used in a vast array of manufactured products as pesticides, herbicides, solvents etc in domestic, agriculture and industrial purposes. However, most of the organohalide compounds are environmentally hazardous, potential carcinogens or mutagens. They are highly insoluble and hydrophobic in nature and therefore get accumulated in fatty tissues of plants, animal food sources 1 . Thus they enter the human body through the food chain and are a potential threat to human life. One of the extensively used and highly contaminating organochlorinated pesticides is Gammaxene 2 . It is classified as the priority pollutant by the US and European Environment Agencies (EPA). Although the developed countries have banned the use of this compound for domestic and agricultural purposes, many developing countries still continue to use it. This compound is also used in small amounts in shampoo and lotions to fight scabies and lice 3,4a . In humans, gammaxene affects the nervous system and also the liver and kidneys causing carcinogenicity, neurological disorders, etc. High insolubility and inertness have made this compound difficult for biodegradation, resulting in the bioaccumulation in human body 4b . This compound has also been found to be poisonous for aquatic life and has been indicated to be a potential carcinogen/mutagen in model organisms 4c . Biodegradation of the highly halogenated compounds have been studied by using certain bacteria and complete dechlorination by reductive dehalogenation was achieved by Shewanella putrefaciens 200 and was proposed to involve reactions of cytochrome c with the pollutants 5 . Reductive dechlorination of certain halogenated hydrocarbons by Methanobacterium thermoauto- trophicum was shown to involve the nickel containing enzyme methyl-coenzyme M reductase 6 . However, there have been no detailed reports on the molecular mechanism of biodegradation of this kind of compounds. Cytochrome P450s, a super-family of b-type heme (Fig. 1A) containing enzymes found in organisms in all domains of life, are involved in C-H activation by molecular oxygen to carry out a large number of metabolic processes like drug metabolism, xenobiotic detoxification as well as in steroid hormone biosynthesis in mammalian tissues 7 . These enzymes are also involved in catalyzing the synthesis of a large number of secondary metabolites in plants and other organisms 8-12 . One of the most extensively studied cytochrome P450, cytochrome P450 cam from the soil bacterium Pseudomonas putida, is found to be