Tyrosine emission in the tryptophanless azurin from Pseudomonas fluorescens

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Abstract
A strain of P. fluorescens contains an azurin with no tryptophan and 2 tyrosines. This protein is interesting because it allows the study of the structure of azurin and the emission of tyrosines in proteins. Comprehensive measurements were done, including spectrophotometric and fluorimetric titration, fluorescence quantum yield, fluorescence polarization and I- quenching. In the Cu-containing protein, almost independent of the Cu ion oxidation, the fluorescence quantum yield is .apprx. 60% of that of the apoprotein. The latter has the remarkable property that its quantum yield is even greater than free tyrosine. The 2 tyrosines in the metalloprotein have different pKa, 10.75 and 12.78, but there is only 1 average pKa, 10.9, in the apoprotein. The polarization of the fluorescence at 310 nm (290-nm excitation) is 0.32 for the metalloproteins and 0.34 for the apoprotein. I- hardly quenches the fluorescence. The 2 tyrosines are inaccessible to the solvent, located in nonpolar environments .gtorsim. 20 .ANG. apart, and not adjacent to the disulfide bridge.