Biophys J 1999 May;76(5):2702-10

Azide reduces the hydrophobic barrier of the bacteriorhodopsin
proton channel.

Steinhoff HJ, Pfeiffer M, Rink T, Burlon O, Kurz M, Riesle J, Heuberger E, Gerwert K, Oesterhelt D

Lehrstuhl fur Biophysik, Ruhr-Universitat Bochum, 44780 Bochum, Germany. hjs@bph.ruhr-uni-bochum.de

The sensitivity of a nitroxide spin label to the polarity of its environment has been used to estimate the hydrophobic
barrier of the proton channel of the transmembrane proton pump bacteriorhodopsin. By means of site-specific
mutagenesis, single cysteine residues were introduced at 10 positions located at the protein surface, in the protein
interior, and along the proton pathway. After reaction with a methanethiosulfonate spin label, the principle values of
the hyperfine tensor A and the g-tensor were determined from electron paramagnetic resonance spectra measured at
170 K. The shape of the hydrophobic barrier of the proton channel is characterized in terms of a polarity index,
DeltaA, determined from the variation of the hyperfine coupling constant Azz. The maximum of the hydrophobic
barrier is found to be close to the retinal chromophore in the proton uptake pathway. The effect of the asymmetric
distribution of charged and polar residues in the proton release and uptake pathways is clearly reflected in the
behavior of the hydrophobic barrier. The presence of azide reduces the barrier height of both the cytoplasmic and
extracellular channels. This finding supports the view of azide and other weakly acidic anions as catalysts for the
formation of hydrogen-bonded networks in proton pathways of proteins.