Q-Band Electron-Nuclear Double Resonance Reveals Out-of-Plane Hydrogen Bonds Stabilize an Anionic Ubisemiquinone in Cytochrome bo3from Escherichia coli.
The respiratory cytochrome bo3ubiquinol 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 O2to water. It is known that SQHis 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, SQHwas investigated with orientation-selective Q-band (∼34 GHz) pulsed1H electron-nuclear double resonance (ENDOR) spectroscopy on fully deuterated cytochrome (cyt) bo3in 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 bo3QHsite 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.
Department of Biochemistry, University of Illinois at Urbana-Champaign , Urbana, Illinois 61801, United States.
Chang
C
Sun
Center for Biophysics and Computational Biology, University of Illinois at Urbana-Champaign , Urbana, Illinois 61801, United States.|Department of Veterinary Clinical Medicine, University of Illinois at Urbana-Champaign , Urbana, Illinois 61801, United States.
Alexander T
AT
Taguchi
Department of Biochemistry, University of Illinois at Urbana-Champaign , Urbana, Illinois 61801, United States.|Center for Biophysics and Computational Biology, University of Illinois at Urbana-Champaign , Urbana, Illinois 61801, United States.
Josh V
JV
Vermaas
School of Chemistry, The University of Manchester , Manchester M13 9PL, U.K.
Nathan J
NJ
Beal
School of Chemistry, The University of Manchester , Manchester M13 9PL, U.K.
Patrick J
PJ
O'Malley
Department of Biochemistry, University of Illinois at Urbana-Champaign , Urbana, Illinois 61801, United States.|Center for Biophysics and Computational Biology, University of Illinois at Urbana-Champaign , Urbana, Illinois 61801, United States.|Beckman Institute, University of Illinois at Urbana-Champaign , Urbana, Illinois 61801, United States.
Emad
E
Tajkhorshid
Department of Biochemistry, University of Illinois at Urbana-Champaign , Urbana, Illinois 61801, United States.|Center for Biophysics and Computational Biology, University of Illinois at Urbana-Champaign , Urbana, Illinois 61801, United States.
Robert B
RB
Gennis
Department of Veterinary Clinical Medicine, University of Illinois at Urbana-Champaign , Urbana, Illinois 61801, United States.
Sergei A
SA
Dikanov
D000838:Anions; D003573:Cytochrome b Group; D003580:Cytochromes / Q000737:chemistry*; D004578:Electron Spin Resonance Spectroscopy; D004583:Electrons; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins / Q000737:chemistry*; D006860:Hydrogen Bonding; D056004:Molecular Dynamics Simulation; D014451:Ubiquinone / Q000031:analogs & derivatives* / Q000737:chemistry
D000838:Anions; D003573:Cytochrome b Group; D003580:Cytochromes; D029968:Escherichia coli Proteins; C083850:cytochrome bo3, E coli; D014451:Ubiquinone; C024989:coenzyme Q10