Azide reduces the hydrophobic barrier of the bacteriorhodopsin proton channel

The sensitivity of a nitroxide spin label to the polarity of its environmen t has been used to estimate the hydrophobic barrier of the proton channel o f the transmembrane proton pump bacteriorhodopsin. By means of site-specifi c mutagenesis, single cysteine residues were introduced at 10 positions loc ated at the protein surface, in the protein interior, and along the proton pathway. After reaction with a methanethiosulfonate spin label, the princip le values of the hyperfine tensor A and the g-tensor were determined from e lectron paramagnetic resonance spectra measured at 170 K. The shape of the hydrophobic barrier of the proton channel is characterized in terms of a po larity index, Delta A, determined from the variation of the hyperfine coupl ing constant A(zz). The maximum of the hydrophobic barrier is found to be c lose to the retinal chromophore in the proton uptake pathway. The effect of the asymmetric distribution of charged and polar residues in the proton re lease and uptake pathways is clearly reflected in the behavior of the hydro phobic barrier. The presence of azide reduces the barrier height of both th e cytoplasmic and extracellular channels. This finding supports the view of azide and other weakly acidic anions as catalysts for the formation of hyd rogen-bonded networks in proton pathways of proteins.