Abstract
The ubisemiquinone stabilized at the Qi-site of the bc1 complex of Rhodobacter sphaeroides forms a hydrogen bond with a nitrogen from the local protein environment, tentatively identified as ring N from His-217. The interactions of 14N and 15N have been studied by X-band (approximately 9.7 GHz) and S-band (3.4 GHz) pulsed EPR spectroscopy. The application of S-band spectroscopy has allowed us to determine the complete nuclear quadrupole tensor of the 14N involved in H-bond formation and to assign it unambiguously to the Nepsilon of His-217. This tensor has distinct characteristics in comparison with H-bonds between semiquinones and Ndelta in other quinone-processing sites. The experiments with 15N showed that the Nepsilon of His-217 was the only nitrogen carrying any considerable unpaired spin density in the ubiquinone environment, and allowed calculation of the isotropic and anisotropic couplings with the Nepsilon of His-217. From these data, we could estimate the unpaired spin density transferred onto 2s and 2p orbitals of nitrogen and the distance from the nitrogen to the carbonyl oxygen of 2.38+/-0.13A. The hyperfine coupling of other protein nitrogens with semiquinone is <0.1 MHz. This did not exclude the nitrogen of the Asn-221 as a possible hydrogen bond donor to the methoxy oxygen of the semiquinone. A mechanistic role for this residue is supported by kinetic experiments with mutant strains N221T, N221H, N221I, N221S, N221P, and N221D, all of which showed some inhibition but retained partial turnover.
Highlights
The bc1 complex functions to oxidize ubihydroquinone (QH2,4 quinol) in
quinone reduction site (Qi-site) Semiquinone in the bc1 Complex implies the existence of an atomic bridge through the H-bond for the transfer of spin density from the SQ
Transferred Spin Density and the Location of the H-bonded Nitrogen Atom—The existence of a nonzero isotropic constant, a, for the interacting imidazole nitrogen indicates that unpaired electron spin density is transferred from the SQ onto this atom
Summary
14N ESEEM—The SQ in frozen solution of native (with the 99.63% of 14N natural abundance) and 15N-labeled bc complex has the same line width ϳ0.8 – 0.85 mT in X-band continuous wave EPR and in field sweep ESE spectra. This spectrum exhibits two lines at frequencies 1.7 and 3.1 MHz. This spectrum exhibits two lines at frequencies 1.7 and 3.1 MHz These two frequencies produce cross-peaks in two-dimensional ESEEM spectra and belong to opposite electron spin manifolds of a 14N nucleus.
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