Abstract
Exchangeable protons in the immediate neighborhood of the semiquinone (SQ) at the Qi-site of the bc1 complex (ubihydroquinone:cytochrome c oxidoreductase (EC 1.10.2.2)) from Rhodobacter sphaeroides have been characterized using electron spin echo envelope modulation (ESEEM) and hyperfine sublevel correlation spectroscopy (HYSCORE) and visualized by substitution of H2O by 2H2O. Three exchangeable protons interact with the electron spin of the SQ. They possess different isotropic and anisotropic hyperfine couplings that allow a clear distinction between them. The strength of interactions indicates that the protons are involved in hydrogen bonds with SQ. The hyperfine couplings differ from values typical for in-plane hydrogen bonds previously observed in model experiments. It is suggested that the two stronger couplings involve formation of hydrogen bonds with carbonyl oxygens, which have a significant out-of-plane character due to the combined influence of bulky substituents and the protein environment. These two hydrogen bonds are most probably to side chains suggested from crystallographic structures (His-217 and Asp-252 in R. sphaeroides). Assignment of the third hydrogen bond is more ambiguous but may involve either a bond between Asn-221 and a methoxy O-atom or a bond to water. The structural and catalytic roles of the exchangeable protons are discussed in the context of three high resolution crystallographic structures for mitochondrial bc1 complexes. Potential H-bonds, including those to water molecules, form a network connecting the quinone (ubiquinone) occupant and its ligands to the propionates of heme bH and the external aqueous phase. They provide pathways for exchange of protons within the site and with the exteriors, needed to accommodate the different hydrogen bonding requirements of different quinone species during catalysis.
Highlights
The quinone1 processing sites of photosynthetic and respiratory electron transfer chains have spawned an extensive literature dealing with mechanism in the context of structure
We present a characterization of the exchangeable protons around SQ in the Qi-site by electron spin echo envelope modulation (ESEEM) and hyperfine sublevel correlation spectroscopy (HYSCORE), visualized by substitution of H2O by 2H2O, in samples of Rhodobacter sphaeroides bc1 complex poised with a substantial population of SQ at the Qi-site
Our results demonstrate the convenience of the ESEEM/HYSCORE approach for studying exchangeable protons around the SQ, without any detectable contribution from substituent protons
Summary
The quinone processing sites of photosynthetic and respiratory electron transfer chains have spawned an extensive literature dealing with mechanism in the context of structure This has been dominated by the two-electron gates of bacterial reaction centers Recent structures at high resolution have suggested that the second quinone processing site of the bc complex, the Qi-site, through which ubiquinone (quinone, Q) is reduced through a two-electron gate, might have interesting dynamic features, albeit on a smaller scale (9 –11). Earlier studies of functional aspects show the same general two-electron gate behavior at the Qi-site as that at the QB-site of bacterial reaction centers but with heme bH of the cytochrome (cyt) b subunit acting as an electron donor [12] In both systems, a relatively stable semiquinone (SQ) intermediate stores one electron from the donor chain and is reduced to quinol by a second electron. We did not detect in the X-band ESEEM spectra any major contributions from other nitrogens in the SQ environment that had comparable unpaired spin density
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