The Semiquinone at the Q_i 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

Abstract

Specific isotopic labeling at the residue or substituent level extends the scope of different spectroscopic approaches to the atomistic level. Here we describe 13C isotopic labeling of the methyl and methoxy ring substituents of ubiquinone, achieved through construction of a methionine auxotroph in Rhodobacter sphaeroides strain BC17 supplemented with l-methionine with the side chain methyl group 13C-labeled. Two-dimensional electron spin echo envelope modulation (HYSCORE) was applied to study the 13C methyl and methoxy hyperfine couplings in the semiquinone generated in situ at the Qi site of the bc1 complex in its membrane environment. The data were used to characterize the distribution of unpaired spin density and the conformations of the methoxy substituents based on density functional theory calculations of 13C hyperfine tensors in the semiquinone of the geometry-optimized X-ray structure of the bc1 complex (Protein Data Bank entry 1PP9) with the highest available resolution. Comparison with other proteins indicates individual orientations of the methoxy groups in each particular case are always different from the methoxy conformations in the anion radical prepared in a frozen alcohol solution. The protocol used in the generation of the methionine auxotroph is more generally applicable and, because it introduces a gene deletion using a suicide plasmid, can be applied repeatedly.

Atomistic determinants of co-enzyme Q reduction at the Qi-site of the cytochrome bc1 complex

“…While this scenario is possible, the other option is that the proton transfers are tightly coupled to separate e − transfers ( Fig. 1B ) as recently suggested by quantum mechanics calculations 16 . Accordingly, the half-protonated SQ ( Fig.…”

“…To address this issue, the bacterial cyt bc 1 complex was studied afresh using explicitly set up MD simulations ( Table 1 ). Because only the anionic SQ has been detected using frozen electron paramagnetic resonance experiments at the Q i -site 12 13 14 15 16 , the substrate has been presumed to acquire both of the electrons (dianionic state) before accepting the two protons concomitantly 17 . While this scenario is possible, the other option is that the proton transfers are tightly coupled to separate e − transfers ( Fig.…”

Can All Major ROS Forming Sites of the Respiratory Chain Be Activated By High FADH2/NADH Ratios?

“…A stable intermediate semiquinone at the Qn site has been studied, see for example, ref. and references therein. As semiquinones have been implicated in ROS production, these discussions are not purely academic.…”

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 À158 mV is shown in Figure 3. It shows an intense unstructured isotropic radical signal centered at g av % 2.0045w ith a peak-to-peak line width of about 0.6 mT assigned to NarGHIbound MSK D with a 2 H-labeled methyl group, hereafter referred to as C 2 H 3 -MSK D .T he EPR peak-to-peak line width of C 2 H 3 -MSK D decreases by % 0.2 mT with respect to that measured for unlabeled MSK D (Figure 3, dashed trace),i ndicating as ignificant contribution of the methyl protons hyperfine coupling to the MSK D EPR linewidth.…”

“…[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).…”