TY - JOUR
T1 - Q-Band Electron-Nuclear Double Resonance Reveals Out-of-Plane Hydrogen Bonds Stabilize an Anionic Ubisemiquinone in Cytochrome bo3 from Escherichia coli
AU - Sun, Chang
AU - Taguchi, Alexander T.
AU - Vermaas, Josh V.
AU - Beal, Nathan J.
AU - O'Malley, Patrick J.
AU - Tajkhorshid, Emad
AU - Gennis, Robert B.
AU - Dikanov, Sergei A.
N1 - Funding Information:
This research was supported by Grants DE-FG02-08ER15960 (S.A.D.) and DE-FG02-87ER13716 (R.B.G.) from the Chemical Sciences, Geosciences and Biosciences Division, Office of Basic Energy Sciences, Office of Sciences, U.S. Department of Energy, and National Center for Research Resources Grants S10-RR15878 and S10-RR025438 for pulsed EPR instrumentation. The computational work at the University of Illinois at Urbana-Champaign was supported by grants from the National Institutes of Health (NIH) to E.T. (U54-GM087519 and P41-RR05969), using resources of the National Energy Research Scientific Computing Center, a DOE Office of Science User Facility supported by the Office of Science of the U.S. Department of Energy under Contract No. DE-AC02-05CH11231. P.J.O. acknowledges the use of computer resources granted by the EPSRC UK national service for computational chemistry software (NSCCS). A.T.T. and J.V.V. gratefully acknowledge past support as NIH trainees of the Molecular Biophysics Training Program (5T32- GM008276). J.V.V. also acknowledges support from the Sandia National Laboratories Campus Executive Program, which is funded by the Laboratory Directed Research and Development (LDRD) Program at Sandia National Laboratories. Sandia is a multiprogram laboratory managed and operated by Sandia Corp., a wholly owned subsidiary of Lockheed Martin Corp., for the U.S. Department of Energys National Nuclear Security Administration under Contract DE-AC04-94AL85000.
Publisher Copyright:
© 2016 American Chemical Society.
PY - 2016/10/11
Y1 - 2016/10/11
N2 - The respiratory cytochrome bo3 ubiquinol oxidase from Escherichia coli has a high-affinity ubiquinone binding site that stabilizes the one-electron reduced ubisemiquinone (SQH), which is a transient intermediate during the electron-mediated reduction of O2 to water. It is known that SQH is stabilized by two strong hydrogen bonds from R71 and D75 to ubiquinone carbonyl oxygen O1 and weak hydrogen bonds from H98 and Q101 to O4. In this work, SQH was investigated with orientation-selective Q-band (∼34 GHz) pulsed 1H electron-nuclear double resonance (ENDOR) spectroscopy on fully deuterated cytochrome (cyt) bo3 in a H2O solvent so that only exchangeable protons contribute to the observed ENDOR spectra. Simulations of the experimental ENDOR spectra provided the principal values and directions of the hyperfine (hfi) tensors for the two strongly coupled H-bond protons (H1 and H2). For H1, the largest principal component of the proton anisotropic hfi tensor Tz′ = 11.8 MHz, whereas for H2, Tz′ = 8.6 MHz. Remarkably, the data show that the direction of the H1 H-bond is nearly perpendicular to the quinone plane (∼70° out of plane). The orientation of the second strong hydrogen bond, H2, is out of plane by ∼25°. Equilibrium molecular dynamics simulations on a membrane-embedded model of the cyt bo3 QH site show that these H-bond orientations are plausible but do not distinguish which H-bond, from R71 or D75, is nearly perpendicular to the quinone ring. Density functional theory calculations support the idea that the distances and geometries of the H-bonds to the ubiquinone carbonyl oxygens, along with the measured proton anisotropic hfi couplings, are most compatible with an anionic (deprotonated) ubisemiquinone.
AB - The respiratory cytochrome bo3 ubiquinol oxidase from Escherichia coli has a high-affinity ubiquinone binding site that stabilizes the one-electron reduced ubisemiquinone (SQH), which is a transient intermediate during the electron-mediated reduction of O2 to water. It is known that SQH is stabilized by two strong hydrogen bonds from R71 and D75 to ubiquinone carbonyl oxygen O1 and weak hydrogen bonds from H98 and Q101 to O4. In this work, SQH was investigated with orientation-selective Q-band (∼34 GHz) pulsed 1H electron-nuclear double resonance (ENDOR) spectroscopy on fully deuterated cytochrome (cyt) bo3 in a H2O solvent so that only exchangeable protons contribute to the observed ENDOR spectra. Simulations of the experimental ENDOR spectra provided the principal values and directions of the hyperfine (hfi) tensors for the two strongly coupled H-bond protons (H1 and H2). For H1, the largest principal component of the proton anisotropic hfi tensor Tz′ = 11.8 MHz, whereas for H2, Tz′ = 8.6 MHz. Remarkably, the data show that the direction of the H1 H-bond is nearly perpendicular to the quinone plane (∼70° out of plane). The orientation of the second strong hydrogen bond, H2, is out of plane by ∼25°. Equilibrium molecular dynamics simulations on a membrane-embedded model of the cyt bo3 QH site show that these H-bond orientations are plausible but do not distinguish which H-bond, from R71 or D75, is nearly perpendicular to the quinone ring. Density functional theory calculations support the idea that the distances and geometries of the H-bonds to the ubiquinone carbonyl oxygens, along with the measured proton anisotropic hfi couplings, are most compatible with an anionic (deprotonated) ubisemiquinone.
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U2 - 10.1021/acs.biochem.6b00669
DO - 10.1021/acs.biochem.6b00669
M3 - Article
C2 - 27622672
AN - SCOPUS:84991251806
VL - 55
SP - 5714
EP - 5725
JO - Biochemistry
JF - Biochemistry
SN - 0006-2960
IS - 40
ER -