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)C-labeled 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.

FIGURE 1.

The three-step PCR technique used to generate linear double-stranded DNA with 500-bp-long homology extensions. In the first step, the upstream 500 bp and the downstream 500 bp of the target chromosomal gene were amplified independently. Each of the UP.R and DOWN.F primers had a short segment homologous to the respective region of the resistance marker. The second step generated the linear DNA from the two homologous 500-bp segments and the resistance marker. In the third step, the linear DNA was transformed into the expression strain and incorporated into the chromosome by homologous recombination.

FIGURE 2.

Contour (A and B) and stacked (C and D) presentations of the HYSCORE spectra of the UQ-8 with ¹³C-labeled methyl and methoxy groups in the Q_H site of uniformly ¹⁵N-labeled wild-type cyt bo₃ (A and C) and in frozen isopropyl alcohol solution (B and D) (magnetic field 346.3 mT (bo₃) and 345.1 mT (isopropyl alcohol), time between first and second pulses τ = 136 ns, microwave frequency 9.711 GHz (bo₃) and 9.682 GHz (isopropyl alcohol)).

FIGURE 3.

Pulsed ENDOR spectra obtained using Mims ENDOR sequence for the ¹³C-labeled UQ-8 in the Q_H site of cyt bo₃ and UQ-8 anion radical in isopropyl alcohol. Length of the π/2 microwave pulse is 48 ns, and the length of the radiofrequency π pulse is 10 μs. The spectra shown represent a sum of seven individual spectra recorded at different τ values varied from initial time τ = 200 ns with the step 24 ns (magnetic field 344.3 mT (bo₃) and 344.5 mT (isopropyl alcohol), microwave frequency 9.668 GHz (bo₃) and 9.663 GHz (isopropyl alcohol)).

FIGURE 4.

Contour (A and B) and stacked (C and D) presentations of the HYSCORE spectra of the Q_H SQ with ¹³C-labeled methyl and methoxy groups in uniformly ¹⁵N-labeled D75E and D75H enzymes (magnetic field 346.2 mT (D75E) and 345.7 mT (D75H), time between first and second pulses τ = 136 ns, microwave frequency 9.706 GHz (D75E) and 9.697 GHz (D75H)).

FIGURE 5.

Comparative view of the lines from methoxy groups in the stacked presentation of the ¹³C HYSCORE spectra of wild-type cyt bo₃ (top) and the D75E (middle) and D75H (bottom) mutants. Corresponding full ¹³C spectra are shown in Figs. 2C and 4 (C and D).

SCHEME 1