The previously described correlation between the ferric spin equilibrium of cytochrome P-450cam and the environmental polarity of tyrosine residues (Fisher et al., 1986) has been further examined with the use of site-directed mutagenesis and active-site affinity reagents. Whereas the wild-type demonstrates an increase in environmental polarity of approximately one tyrosine residue, the mutant protein Y96F, in which Tyr-96 has been changed to Phe-96, demonstrates a lack of spin-state-dependent change in the second-derivative ultraviolet absorption spectrum. This suggests that the active-site Tyr-96 serves as a ultraviolet spectroscopic probe which can be utilized to determine the relative degree of water access to the active site for various substrate/protein complexes. The affinity reagent isobornyl mercaptan has been used to demonstrate the utility of this probe in determining the active-site polarity when substrate analogues are bound at the active site. In addition, the sensitivity of Tyr-96 to environmental polarity has been used to demonstrate that the product/enzyme complex, formed with 5-exo-hydroxycamphor, may be associated with increased water access to the heme iron. This may provide a means for turning off electron transfer when the product, instead of the substrate, is bound at the active site.
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