Hydrogen Bonds between Nitrogen Donors and the Semiquinone in the Qi-site of the bc1 Complex

Abstract

The ubisemiquinone stabilized at the Q i -site of the bc 1 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 14 N and 15 N have been studied by X-band (ϳ9.7 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 14 N involved in H-bond formation and to assign it unambiguously to the N ⑀ of His-217. This tensor has distinct characteristics in comparison with H-bonds between semiquinones and N ␦ in other quinone-processing sites. The experiments with 15 N showed that the N ⑀ 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 N ⑀ 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 ؎ 0.13 Å . 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.The bc 1 complex (ubihydroquinone:cytochrome c oxidoreductase, or complex III of the mitochondrial respiratory chain) functions to oxidize ubihydroquinone (QH 2 , 4 quinol) in the membrane and reduce cytochrome c (or cytochrome c 2 in bacteria) in the aqueous phase, using the redox work to transport 2H ϩ per electron across the membrane (1-4). In the photosynthetic bacterium Rhodobacter sphaeroides, the bc 1 complex completes the photosynthetic chain containing the photochemical reaction center, which provides the substrates for the complex following photoactivation (5, 6). The Q-cycle mechanism is a well tested model to account for the function of the bc 1 complex (7-9), but several details are still controversial. The Q-cycle involves separate catalytic sites on opposite sides of the membrane for oxidation of QH 2 and reduction of ubiquinone (Q, quinone). The work function is provided by a bifurcation of electron transfer at the quinol-oxidizing site (the Q osite). The first electron is transferred to a high potential chain (the Rieske iron-sulfur protein, cytochrome c 1 , and cytochrome c), and the second to a low potential chain, consisting of heme b L and heme b H in cytochrome b, which delivers electrons to the quinone reduction site (Q i -site). Some of the work from the first electron transfer is stored in the strong reducing potential of an unstable semiquinone intermediate formed at the Q o -site. This provides the driving force for reduction of Q, or of ubisemiquinone (SQ), in the Q i -site, and for generation of the p...

Characterization of the Semiquinone Radical Stabilized by the Cytochrome aa3-600 Menaquinol Oxidase of Bacillus subtilis

“…(40). On the other hand, this coupling constant is larger than the K values reported for either the deprotonated or protonated nitrogens of the imidazole residue (36). That rules out histidine as a hydrogen bond partner to the SQ of cyt aa 3 -600.…”

“…This is due to the influence of the nuclear quadrupole interaction (3,4,6,36 (3,36), suggesting that more than one nitrogen interacts with the SQ, accompanied by the transfer of unpaired spin density onto their nuclei.…”

Electronic Connection Between the Quinone and CytochromecRedox Pools and Its Role in Regulation of Mitochondrial Electron Transport and Redox Signaling

“…This scheme is supported by the observation that USQ i is in anionic form (USQ i Ϫ ) (76,143). Crystal structures revealed two regions rich in protonable groups of amino acid side chains and water molecule paths for consideration as proton paths linking the catalytic site with the membrane surface (129,153).…”

“…Mutagenic studies and EPR spectroscopy identified conservative residues responsible for binding and stabilization of quinone and USQ i (76,112,120,143 (149,242).…”

Resolving the EPR Spectra in the Cytochrome bc 1 Complex from Saccharomyces cerevisiae

“…More recently similar experiments have been performed on the bacterial bc 1 complex from Rhodobacter (Rb.) sphaeroides and a direct binding to histidine has been proposed [23,24]. When applying EPR spectroscopy to study electron-transfer intermediates in mitochondrial or bacterial respiratory chains, it is important to realize that presence of several paramagnetic species is a common occurrence.…”