Electron Transfer Induces Side-Chain Conformational Changes of Glutamate-286 from Cytochrome bo₃

Abstract

Heme-copper oxidases have two putative proton channels, the so-called K-channel and the membrane-spanning D-channel. The latter contains a number of polar groups with glutamate-286 located in its center, which could-together with bound water-contribute to a transmembrane hydrogen-bonded network. Protonation states of carboxyl groups from cytochrome bo3 of Escherichia coli were studied by redox Fourier transform infrared (FTIR) difference spectroscopy. A net absorbance increase in the carboxyl region was observed upon reduction. The band signature typically found in heme-copper oxidases comprises an absorbance decrease (reduced-minus-oxidized difference spectra) at 1745 cm-1 and increase at 1735 cm-1. No significant changes in the carboxyl region were found in the site-specific mutants D135E and D407N. The difference bands were lacking in redox spectra of mutants at position 286; they 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 the carboxyl group an orientation toward the interior of the D-channel for the 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 CuB metal centers.

Structural elements involved in electron-coupled proton transfer in cytochrome c oxidase

“…An analysis of the X-ray structure supports the conclusion that E(I-286) is protonated at neutral pH, forming a hydrogen bond with the carbonyl oxygen of M(I-107) [11]. This observation is also consistent with results from experiments using FT-IR spectroscopy, showing high pK a values (>9) of the corresponding glutamate in haemcopper oxidases from different species [52,53].…”

Glutamic acid 242 is a valve in the proton pump of cytochrome c oxidase

“…While the up and down configurations of the protonated Glu-242 have approximately equal probabilities when heme a is reduced and the binuclear site is oxidized (P M state), moving the electron from heme a to the binuclear site to yield the P R state changed the distribution to an Ϸ20-fold preference of the down state. This finding may explain observations by infrared spectroscopy, where the characteristic absorption of the protonated Glu-242 near 1,740 cm Ϫ1 is shifted in a manner that depends on the redox state of heme a (37)(38)(39)(40)(41). Fig.…”

Recent advances in the electrochemistry and spectroelectrochemistry of membrane proteins

“…This residue is the key proton transporter within the proton pump (Brzezinski et al , 2008 ). Analysis of the infrared signal was confirmed by groups working on the same enzyme from different organisms L ü bben et al, 1999 ).…”