Selective nitration of tyrosines-26 and -64 in bacteriorhodopsin with tetranitromethane

Nitration of tyrosine-26 at pH 9.0 in bacteriorhodopsin does not change its absorption spectrum but lowers the apparent pK of the alkaline transition to a blue-shifted chromophore from about pH 12.0 to 10.6. This effect is reversed by reducing the nitrotyrosine-26 to aminotyrosine which demonstrates that the protonation state of tyrosine-26 and the alkaline chromophore transition are correlated. Nitration of tyrosine-64 resulted in a shift of the purple complex from 570 to 535 nm at neutral pH. The alkaline transition pK of such a nitrated membrane was below 10 but was clearly independent of the protonation state of tyrosine-64 because it is not reversed by reduction of the nitrotyrosine. Nitrotyrosine-26 showed spectral properties similar to L-nitrotyrosine in aqueous environment while nitrotyrosine-64 showed only a 360-nm absorbance in the apomembrane but not in the retinal-containing membrane. Both tyrosines are accessible to water-soluble reagents.