Properties of the tryptophan residue in rabbit liver microsomal cytochrome P-450 isozyme 2 as determined by fluorescence

Abstract

Cytochrome P-450 isozyme 2 from rabbit liver microsomes fluoresces upon excitation at 295 nm due to the single tryptophyl residue (Trp 121) in the protein. The fluorescence spectrum, which is not altered by the presence of phospholipid or substrates, has a maximum at 335 nm, which suggests that the environment of the residue is hydrophobic. The fluorescence intensity decreases linearly with increase of specific content of the cytochrome preparations, and the holoenzyme was estimated to exhibit, at most, 6% as much fluorescence as the apoenzyme. This indicates that the fluorescence of the tryptophan is quenched by energy transfer to the heme. The distance between the tryptophyl residue and the heme was estimated to be less than 40 Å. From enhancement of the fluorescence by methanol and ethanol, 30 to 50% of the Trp residue was found to be accessible to these solvents. On the other hand, the accessibility to iodide and cesium ions, as estimated by quenching effects, is less than 14%. From such evidence, the tryptophyl residue is believed to be partly buried. Since Trp 121 is conserved at or near the same position in all mammalian P-450's so far sequenced, the results obtained may be applicable to these related cytochromes as well.