Electron transfer induces side-chain conformational changes of glutamate-286 from cytochrome bo3

Heme-copper oxidases have two putative proton channels, the so-called K-cha nnel and the membrane-spanning D-channel. The latter contains a number of p olar groups with glutamate-286 located in its center, which could-together with bound water-contribute to a transmembrane hydrogen-bonded network. Pro tonation states of carboxyl groups from cytochrome bo(3) of Escherichia col i were studied by redox Fourier transform infrared (FTIR) difference spectr oscopy. A net absorbance increase in the carboxyl region was observed upon reduction. The band signature typically found in heme-copper oxidases compr ises an absorbance decrease (reduced-minus-oxidized difference spectra) at 1745 cm(-1) and increase at 1735 cm(-1). No significant changes in the carb oxyl region were found in the site-specific mutants D135E and D407N. The di fference bands were lacking in redox spectra of mutants at position 286; th ey could clearly be related to Glu-286. In wild-type oxidase, the pK of Glu -286 appears to be higher than 9.8. Upon solvent isotope exchange from H2O to D2O, the band at 1745 cm(-1) shifts more readily than the one at 1735 cm (-1), indicating dissimilar accessibility of the carboxyl side chain to the hydrogen-bonded network in both redox states, The data are consistent with a redox-triggered conformational change of Glu-286, which attributes to th e carboxyl group an orientation toward the interior of the D-channel for th e oxidized form. The change of Glu-286 is retained in cyanide complexes of cytochrome bo3 and of cytochrome c oxidase: therefore it should be related to oxidoreduction of the heme b and/or Cu-B metal centers.