Exploring by Pulsed EPR the Electronic Structure of Ubisemiquinone Bound at the QH Site of Cytochrome bo3 from Escherichia coli with in Vivo 13C-Labeled Methyl and Methoxy Substituents

Abstract

The cytochrome bo 3 ubiquinol oxidase from Escherichia coli resides in the bacterial cytoplasmic membrane and catalyzes the two-electron oxidation of ubiquinol-8 and four-electron reduction of O 2 to water. The one-electron reduced semiquinone forms transiently during the reaction, and the enzyme has been demonstrated to stabilize the semiquinone. The semiquinone is also formed in the D75E mutant, where the mutation has little influence on the catalytic activity, and in the D75H mutant, which is virtually inactive. In this work, wild-type cytochrome bo 3 as well as the D75E and D75H mutant proteins were prepared with ubiquinone-8 13 C-labeled selectively at the methyl and two methoxy groups. This was accomplished by expressing the proteins in a methionine auxotroph in the presence of L-methionine with the side chain methyl group 13 C-labeled. The 13 Clabeled quinone isolated from cytochrome bo 3 was also used for the generation of model anion radicals in alcohol. Two-dimensional pulsed EPR and ENDOR were used for the study of the 13 C methyl and methoxy hyperfine couplings in the semiquinone generated in the three proteins indicated above and in the model system. The data were used to characterize the transferred unpaired spin densities on the methyl and methoxy substituents and the conformations of the methoxy groups. In the wild type and D75E mutant, the constraints on the configurations of the methoxy side chains are similar, but the D75H mutant appears to have altered methoxy configurations, which could be related to the perturbed electron distribution in the semiquinone and the loss of enzymatic activity. Ubiquinone (UQ)3 functions as a membrane-soluble twoelectron carrier. It is often found as a cofactor in many respiratory and photosynthetic complexes and plays an important role in biological oxidation-reduction reactions. Many of these reactions consist of one-electron transfer steps and involve the one-electron reduced ubisemiquinone species as the intermediates. Knowledge of how the semiquinone (SQ) radicals are stabilized in these complexes is critical to understand their reaction mechanisms. The quinone binding sites located in the respiratory and photosynthetic enzymes are significantly different (1), and each protein fine tunes the properties of the bound quinone cofactor by providing a unique environment in order to accomplish its own electron transfer process (2). The well characterized quinone binding sites include those from bacterial photosynthetic reaction centers (3), cytochrome bo 3 ubiquinol oxidase (cyt bo 3 ) from Escherichia coli (4), cytochrome bc 1 complexes (5), photosystems I and II, and some others (6 -9).A SQ can bind within a protein site in a manner that favors either the neutral (QH ⅐ ) or anionic (Q . ) form or intermediate states with partial charge remaining on the SQ. The proton locations along the hydrogen bonds to the quinone carbonyls determine the net charge on the SQ. The formation of hydrogen bonds of different strengths to the quinone oxygens usually leads to an asym...

Q-Band Electron-Nuclear Double Resonance Reveals Out-of-Plane Hydrogen Bonds Stabilize an Anionic Ubisemiquinone in Cytochromebo3fromEscherichia coli

“…18 The distances and angles above allow us to conclude that the SQ H stabilized in cyt bo 3 is the anionic SQ. In addition, the significant asymmetry of the spin density distribution in SQ H revealed by 1 H and 13 C 5′-methyl couplings 21–26 and the 13 C hfi tensors for C 1 and C 4 24 are consistent with this H-bond geometry. The unusual characteristics of H1 result from the strong ~70° out of plane H-bond deviation.…”

“…Calculated hfi couplings can also be compared with previous experimental determinations of 1 H (10–11 MHz) 21–25 and 13 C (−6.1 MHz) 26 isotropic constants for the 5′-methyl group and 13 C anisotropic hfi tensors and isotropic constants for C 1 (−8.9, −17.3, 26.1) MHz, a =4.7 MHz and C 4 (−7.9, −11.3, 19.3) MHz, a = 0.9 MHz carbonyl atoms in SQ H , respectively. 24 All these characteristic hfi couplings have been measured in several studies of SQs and indeed are the principal indicator that the spin density distribution in the SQ H is highly asymmetric compared with a practically symmetric distribution in the anion radical generated in water or alcohol solutions.…”

“…20 In addition, weak hfi couplings with the side-chain and peptide nitrogens from R71, H98, and Q101 were resolved, thus providing a full view of the spin density transfer from SQ H to the nearest residues. 19,20 These data are supported by the ~10–11 MHz isotropic coupling with the 5′-methyl substituent protons (about twice the value for the SQ in alcohol solutions), 21–25 and 13 C couplings in 5′-methyl 26 and carbonyls, 24. all which indicate significant asymmetry in the distribution of the unpaired spin density.…”

The Semiquinone at the Qi Site of the bc1 Complex Explored Using HYSCORE Spectroscopy and Specific Isotopic Labeling of Ubiquinone in Rhodobacter sphaeroides via 13C Methionine and Construction of a Methionine Auxotroph

“…5,31 However, as noted above, these data are currently not available for the SQ i in the Q i site, because approaches for selective isotopic labeling of proteins and cofactors have not been developed in Rb. sphaeroides .…”

Probing the Menasemiquinone Binding Mode to Nitrate Reductase A by Selective 2H and 15N Labeling, HYSCORE Spectroscopy, and DFT Modeling

“…[42] In addition, as imilar approach using selectively 13 Clabeled methionines allowed selective 13 Ce nrichmento ft he methyl and methoxy substituents of ubisemiquinoneb ound at the cyt bo 3 Q H site from E. coli or at the bc 1 complex Q i site from Rhodobacter sphaeroides. [52,53] NarGHI-enriched inner membrane vesicles were purified from the methionine-deficient strain mentioned above, titrated, and studied by EPR spectroscopy.T he cw EPR spectrumo fasample poiseda t Figure 2. Contour presentationsoft he low-frequency part of representative HYSCORE spectrao fMSK D by using eitheru nenriched NarGHI (A),u niform 15 Ne nrichment of NarGHI (B), selectively 15 N-labeled NarGHI on His Nd (C), or selectively 15 N-labeled NarGHI on LysNz (D).…”