Vibrational Modes of Ubiquinone in Cytochrome bo₃ from Escherichia coli Identified by Fourier Transform Infrared Difference Spectroscopy and Specific ¹³C Labeling

Abstract

In this study we present the infrared spectroscopic characterization of the bound ubiquinone in cytochrome bo(3) from Escherichia coli. Electrochemically induced Fourier transform infrared (FTIR) difference spectra of DeltaUbiA (an oxidase devoid of bound ubiquinone) and DeltaUbiA reconstituted with ubiquinone 2 and with isotopically labeled ubiquinone 2, where (13)C was introduced either at the 1- or at the 4-position of the ring (C=O groups), have been obtained. The vibrational modes of the quinone bound to the discussed high-affinity binding site (Q(H)) are compared to those from the synthetic quinones in solution, leading to the assignment of the C=O modes to a split signal at 1658/1668 cm(-)(1), with both carbonyls similarly contributing. The FTIR spectra of DeltaUbiA reconstituted with the labeled quinones indicate an essentially symmetrical and weak hydrogen bonding of the two C=O groups from the neutral quinone with the protein and distinct conformations of the 2- and 3-methoxy groups. Perturbations of the vibrational modes of the 5-methyl side groups are discussed for a signal at 1452 cm(-)(1). Only negligible shifts of the aromatic ring modes can be reported for the reduced and the protonated form of the quinone. Alterations of the protein upon quinone binding are reflected in the electrochemically induced FTIR difference spectra. In particular, difference signals at 1640-1633 cm(-)(1) and 1700-1670 cm(-)(1) indicate variations of beta-sheet secondary structure elements and loops, bands at 1706 and 1678 cm(-)(1) are tentatively attributed to individual amino acids, and a difference signal a 1540 cm(-)(1) is discussed to reflect an influence on C=C modes of the porphyrin ring or on deprotonated propionate groups of the hemes. Further tentative assignments are presented and discussed. The (13)C labeling experiments allow the assignment of the vibrational modes of a bound ubiquinone 8 in the electrochemically induced FTIR difference spectra of wild-type bo(3).

The Conformational Changes Induced by Ubiquinone Binding in the Na+-pumping NADH:Ubiquinone Oxidoreductase (Na+-NQR) Are Kinetically Controlled by Conserved Glycines 140 and 141 of the NqrB Subunit

“…In either case the positive peaks in the WT-minus-G140A double difference spectrum at 1676 and 1657 cm Ϫ1 could represent a reduction in the quantity of bound ubiquinone in the NqrB-G140A mutant. However, there is no difference in other peaks previously established to correspond to ubiquinone, such as 1288 and 1265 cm Ϫ1 (40). Thus the data might indicate that the redox-driven conformational changes in these mutants are altered in comparison to wild-type Na ϩ -NQR.…”

“…The corresponding spectra in the absence of CoQ-1 are displayed in gray. Previously, similar experiments were carried out for the photosynthetic reaction center (41) and cytochrome bo 3 (40) in the presence of ubiquinone; therefore, modes corresponding to ubiquinone are well known and can be assigned. Unbound ubiquinone in aqueous solution is known to have a broad positive peak at 1652 cm Ϫ1 with a shoulder at 1664 cm Ϫ1 in the oxidized-minusreduced difference spectra that is due to a redox-induced change in the (CAO) stretch.…”

“…This decrease in amplitude could be due to a restriction in conformational flexibility in response to the mutation to a less flexible residue. However, (CAO) ubiquinone peaks are also expected to fall in the region of 1660 -1670 cm Ϫ1 (40). In this region there are two peaks that could contain contributions from ubiquinone in the wild-type spectrum (Fig.…”

“…6A): at 1660 and 1673 cm Ϫ1 . These two peaks could describe two ubiquinone molecules bound to wild-type Na ϩ -NQR, or alternatively, they could contain contributions from only one ubiquinone molecule where the two carbonyl groups are bound in a different manner, splitting the signal into Peaks from the oxidized-minus-reduced FTIR difference spectra of the wild-type Na ؉ -NQR (WT), the NqrB-G140A mutant, and the NqrB-G141V mutant for the potential step between ؊620 and ؉200 mV in the absence or presence of 1:1 ubiquinone-1 two parts (40). In either case the positive peaks in the WT-minus-G140A double difference spectrum at 1676 and 1657 cm Ϫ1 could represent a reduction in the quantity of bound ubiquinone in the NqrB-G140A mutant.…”

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

“…22 Variant cytochrome bo 3 ubiquinol oxidase from E. coli containing one equivalent of bound UQ 8 was purified in n-dodecyl-b-D-maltoside according to the method described previously, 28 and concentrated to approximately 0.1 mM in 100 mM K-Phosphate buffer at pH 8. The presence or absence of ubiquinone was confirmed by FTIR spectroscopy as reported in 31 (data not shown).…”