Q-Band ENDOR (Electron Nuclear Double Resonance) of the High-Affinity Ubisemiquinone Center in Cytochrome bo₃ from Escherichia coli

Abstract

Electron nuclear double resonance (ENDOR) was performed on the protein-bound, stabilized, high-affinity ubisemiquinone radical, QH*-, of bo3 quinol oxidase to determine its electronic spin distribution and to probe its interaction with its surroundings. Until this present work, such ENDOR studies of protein-stabilized ubisemiquinone centers have only been done on photosynthetic reaction centers whose function is to reduce a ubiquinol pool. In contrast, QH*- serves to oxidize a ubiquinol pool in the course of electron transfer from the ubiquinol pool to the oxygen-consuming center of terminal bo3 oxidase. As documented by large hyperfine couplings (>10 MHz) to nonexchangeable protons on the QH*- ubisemiquinone ring, we provide evidence for an electronic distribution on QH*- that is different from that of the semiquinones of reaction centers. Since the ubisemiquinone itself is physically nearly identical in both QH*- and the bacterial photosynthetic reaction centers, this electronic difference is evidently a function of the local protein environment. Interaction of QH*- with this local protein environment was explicitly shown by exchangeable deuteron ENDOR that implied hydrogen bonding to the quinone and by weak proton hyperfine couplings to the local protein matrix.

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

“…Additional hyperfine structure (also better resolved in the sample prepared in 2 H 2 O) can be seen in the area around g yy ; however, its complete resolution would require experiments at microwave frequencies ϳ95 GHz or higher. The components of the g-tensor, determined from the Q-band spectra of cyt aa 3 -600 SQ prepared in H 2 O and 2 H 2 O, are within the range previously reported for various SQs in model systems and in proteins (30,33,34). A Q-band spectrum with a similar shape was previously reported for the semiquinone intermediate stabilized in the membrane-bound subunit NarI of nitrate reductase (NarGHI) from E. coli (35).…”

Occurrence of a Bound Ubiquinone and Its Function in Escherichia coli Membrane-bound Quinoprotein Glucose Dehydrogenase

“…Reported EPR spectra of the PQQ radicals in MDH (42), soluble glucose dehydrogenase (43), and ADH II (44) have features similar to that shown in Fig. 2, whereas EPR spectra of ubisemiquinone radicals in cytochrome-bo 3 oxidase (34,45) are apparently different, exhibiting hyperfine splitting because of the 5-methyl hydrogen nuclei. On the other hand, estimation of the signal intensity, using the spectra from PQQ-⌬UbiA mGDH as a reference, has revealed that 60 -80% of PQQ is converted to the radical form.…”

Elucidating mechanisms in haemcopperoxidases: The high-affinity QHbinding site in quinol oxidase as studied by DONUT-HYSCOREspectroscopy and density functional theory

“…1C, ii). Spectral simulation (dotted line) clearly reveals two distinct methyl HFC's with (F) 14 N-DONUT-HYSCORE spectrum (absolute value mode) of Q _ H in wt-QOX (D) and Q _ H in D75H-QOX (F). Again in this experiment the time between the first two microwave pulses was s ¼ 132 ns and the times t 1 (between the second and third microwave pulses) and t 2 (between the fourth and fifth microwave pulses) were incremented in steps of 16 ns from their initial values and 512 points were recorded in each dimension.…”