Identification of the Nitrogen Donor Hydrogen Bonded with the Semiquinone at the QH Site of the Cytochrome bo3 from Escherichia coli

E. coli cytochrome (cyt) bo3 ubiquinol oxidase catalyzes the two-electron oxidation of ubiquinol and the four-electron reduction of O2 to water. The enzyme contains three redox-active metal centers: a low spin heme b, which is involved in quinol oxidation, and the heme o3/CuB bimetallic center, which is the site where O2 binds and is reduced to water. The ubiquinol oxidation occurs with a semiquinone (SQ) intermediate in an overall reaction that releases two protons to the periplasm. The enzyme contains two Q sites1-6: a low affinity site (QL), which is equilibrated with the quinone pool in the membrane and functions as the substrate (QH2) binding site, and a high affinity (QH) site, from which Q is not readily removed, and which stabilizes a SQ.7-10 The QH site quinone appears to function as a tightly bound cofactor, similar to the QA site of the reaction centers. The X-ray structure of cyt bo311 does not contain any bound quinone, but mutational substitutions of R71, D75, H98, and Q101 modulate the properties of the QH site, forming the basis of a model of QH binding site (see Supporting Info).2-4,11 The interaction of the SQ with the protein environment in cyt bo3 has been studied by pulsed EPR spectroscopy. X-band ESEEM data show that there is one H-bond to the QH SQ from a nitrogen donor.5,6,12,13 The speculated identification of this nitrogen has been based on the quadrupole coupling constant (qcc) determined from the ESEEM spectra.5,6,12,13 Its value, K=e2qQ/4h=0.93 MHz, most closely corresponds to the nitrogen from an NH or NH2 group.12,13 This value is ∼10% larger than the qcc for the peptide amide nitrogen and significantly exceeds the qcc of the protonated nitrogens in histidine. Hence, the most likely candidates for the H-bond donor are the nitrogens from the side chains of R71 or Q101, though a peptide backbone nitrogen cannot be ruled out completely. To overcome the existing uncertainties and to identify directly the H-bonded nitrogen, we employed 15N selective labeling in different residues. Proteins were labeled as follows: 1) 15N uniformly labeled Arg; 2) 15N uniformly labeled His; 3) Gln with 15N only in the Ne position; 4) Arg with 15N only in the two Nη positions; 5) Arg with 15N only in the peptide nitrogen (Scheme 1). The 15N labeling procedures, the preparation of EPR samples and generation of the SQ are described in Supporting Info. To resolve the interaction of the SQ with 14N and 15N nitrogens we used X-band three-pulse ESEEM and 2D ESEEM (HYSCORE). Scheme 1 The X-band three-pulse 14N ESEEM spectrum of the SQ in wild-type cyt bo3 has been described in detail previously.5,6,12,13 It consists of three-intensive narrow lines: νo=0.95 MHz, ν− =2.32 MHz, and ν+=3.27 MHz, where ν+ =νo + ν−. There is also a less intensive and broader line at frequency νdq ∼5.1-5.2 MHz (see Supporting Info). This spectrum is typical for a single 14N at near cancellation conditions (|νN – A/2|∼0; νN – 14N Zeeman frequency, ∼1.07 MHz in X-band, A – hyperfine coupling). The narrow peaks are assigned to the three nuclear quadrupole resonance frequencies, and broader line is the frequency of the double-quantum transition νdq from the opposite manifold. The spectroscopic parameters determine the qcc K=0.93 MHz, the asymmetry parameter η=0.51, and the hyperfine coupling A = 1.8 MHz for this nitrogen.5,6,12,13 The corresponding HYSCORE spectrum of the SQ in wild-type bo3 (Fig. 1a) exhibits cross-peaks correlating νo, ν−, and ν+ with νdq, thus indicating that they belong to different manifolds. The most intensive cross-peaks in Fig. 1a are from the (ν+,νdq) correlations. There are also three intensive peaks at the diagonal points corresponding to νo, ν−, and ν+. Figure 1 14N and 15N HYSCORE spectra in contour presentation of the SQ in the QH-site of the wild-type bo3 oxidase (a), bo3 with 15N labeled Nηs in R71 (b), uniformly 15N-labeled H98 (c), uniformly 15N-labeled R71 (d). Magnetic field, time τ, and ... Specifically, the 15N-labeled proteins were examined in order to identify the nitrogen responsible for the 14N features observed with wild-type protein. It is assumed that only R71, Q101 and H98 are significant in interpreting the results from 15N-labeled Arg, Gln and His residues, respectively. Fig. 1b shows the spectrum of bo3 with 15N-labeled Nη in R71. In addition to the unchanged 14N features, this spectrum resolves two new weak cross-peaks from 15N centered around a diagonal point with a 15N Zeeman frequency 15νN∼1.5 MHz with coordinates (1.58, 1.43) MHz corresponding to the hyperfine coupling 15A=0.15 MHz. The spectrum of the bo3 with 15N labeled Np of R71 did not show any resolved peaks from 15N. A peak of very low intensity located at the diagonal point (15νN,15νN) was observed for the bo3 with labeled Ne of Q101, thus indicating very weak dipolar interaction between the unpaired electron and a distant 15N nucleus. The spectrum of the bo3 with uniformly 15N-labeled H98 (Fig. 1c) also shows the peak with a maximum at the diagonal point (15νN,15νN). However, it is accompanied by extended shoulders with weakly resolved maxima corresponding to couplings of ∼0.3 and 0.6 MHz. This line can be produced by the interactions with up to three 15Ns, and more specific labeling will be needed to resolve the exact coupling from each nitrogen of this residue. The spectra of these samples have clearly shown that labeled nitrogens in each of the residues R71 (except Np), Q101 and H98 are located in close proximity of the paramagnetic SQ and some of them even carry a small fraction of the unpaired spin density producing isotropic hyperfine splittings of ∼0.1 to 0.6 MHz. However, none of these 15N-labeled positions is responsible for the X-band 14N ESEEM features of the wild-type protein which would result in a significantly larger 15N hyperfine coupling, 15A∼2.5 MHz (recalculated from A∼1.8 MHz for 14N). A dramatic change of the ESEEM spectra, accompanied by the complete disappearance of the 14N peaks, is observed in the bo3 with uniformly 15N-labeled R71 (Fig 1d). The HYSCORE spectrum of the SQ in this protein contains two intense cross-peaks (Ne) from 15N with a maximum at (2.74, 0.34) MHz corresponding to the coupling 15A=2.4 MHz, as well as weak features similar to ones observed for R71 with 15Nηs (Fig. 1b). The extended shape of the Ne peaks results from the anisotropic hyperfine interaction with the average perpendicular component of the tensor T∼0.4 MHz. Taking into account that R71, selectively labeled in the Nη and Np positions, does not result in features correlating to a large hyperfine coupling, it can be definitively concluded that the Ne of R71 is an H-bond donor to the carbonyl oxygen of the SQ. The unpaired spin density fraction of ∼1.2·10-3 is transferred onto the Ne nucleus through the H-bond bridge (see Supporting Info). Spin density delocalization continues further through Ne and reaches at least one Nη, which is separated by two bonds from the Ne in the side-chain, producing the resolved coupling 15A∼0.15 MHz. These experiments also provide evidence that some unpaired spin density (15A coupling up to 0.6 MHz) is transferred to the nitrogens of H98, suggesting its involvement in an H-bond with a larger O-H bond distance or with a less favorable configuration of the spin density transfer than the H-bond with the Ne of R71. The weakly coupled nitrogens with 15A<0.6 MHz (14A<0.43 MHz) are far away from the cancellation condition and, as a result, do not produce resolved contributions to the X-band 1D and 2D 14N ESEEM spectra. The evidence also supports a weak interaction of the SQ with the side chain nitrogen of Q101. In summary, the experiments with the QH site SQ in the series of selectively 15N labeled bo3 oxidases have directly identified the Ne of R71 as the H-bond donor responsible for the features observed in the X-band 14N ESEEM of wild-type bo3. In addition, selective 15N labeling has allowed us for the first time to probe a distribution of the unpaired spin density on the side-chain nitrogens from the residues around the SQ in the quinone binding site. These results form a basis for more advanced theoretical analysis of the protein-SQ interaction in the QH site, using the model of the neutral radical indicated by the proton hyperfine couplings12,13, in conjunction with other available magnetic resonance data.14