Redox-induced Protein Structural Changes in Cytochrome bo Revealed by Fourier Transform Infrared Spectroscopy and [13C]Tyr Labeling

Abstract

Cytochrome bo is a heme-copper terminal ubiquinol oxidase of Escherichia coli under highly aerated growth conditions. Tyr-288 present at the end of the K-channel forms a C ⑀ -N ⑀ covalent bond with one of the Cu B ligand histidines and has been proposed to be an acid-base catalyst essential for the O-O bond cleavage at the Oxyto-P transition of the dioxygen reduction cycle (Uchida, T., Mogi, T., and Kitagawa, T. Cytochrome bo is a four-subunit ubiquinol oxidase in the aerobic respiratory chain of Escherichia coli and belongs to the heme-copper terminal oxidase superfamily (1-3). Subunit I binds all four redox centers, the high affinity ubiquinone binding site (Q H 1 ), low spin heme b, high spin heme o, and Cu B ; the latter two centers form the heme o-Cu B binuclear center. Quinols are oxidized at the low affinity quinol oxidation site (Q L ) in subunit II, and electrons are sequentially transferred through Q H and heme b to the heme o-Cu B binuclear metal center, where dioxygen reduction takes place (4 -10). The two-electron oxidation of ubiquinol-8 at the periplasmic side of the cytoplasmic membrane is coupled to the four-electron reduction of dioxygen at the cytoplasmic side. Accordingly, four chemical protons are apparently translocated from the cytoplasm to the periplasm, generating an electrochemical proton gradient across the membrane. In addition, the enzyme can vectorially translocate four other protons per dioxygen reduction by a pump mechanism (11). Mutagenesis (12-16) and x-ray crystallographic (17-22) studies on the heme-copper terminal oxidases suggest that the D-and K-channels in subunit I are operative during redox-coupled proton pumping (Fig. 1A). Iwata et al. (18) identified two putative proton channels in cytochrome c oxidase from Paracoccus denitrificans, the D-channel characterized by a conserved Asp residue at the entry site and the K-channel characterized by a conserved Lys residue in the middle of the channel. They proposed that the D-channel translocates four pumped protons, whereas the K-channel delivers four chemical protons to the binuclear center (18). However, it is now assumed that the K-channel delivers one or two chemical protons to the binuclear center at the initial reductive phase of dioxygen reduction and that the D-channel translocates all other chemical and pumped protons (12,(25)(26)(27)(28).In the vicinity of the binuclear center, Tyr-288 (the E. coli cytochrome bo numbering: Tyr-280 in the soil bacterium P. denitrificans and Tyr-244 in bovine cytochrome c oxidase) is highly conserved in the SoxMtype terminal oxidase and has been proposed to be involved in dioxygen reduction (25, 26, 29 -32). Upon two-electron reduction of the enzyme, two chemical protons are taken up from the cytoplasm to compensate for an increased negative charge at the binuclear center (12,33), and at least the first proton is delivered to the binuclear center by Tyr-288 at the end of the K-channel (27). If deprotonated at the oxidized (O) state (20), Tyr-288 serves as one of proton acceptors (26). ...

Characterization of the Phenoxyl Radical in Model Complexes for the CuBSite of CytochromecOxidase: Steady-State and Transient Absorption Measurements, UV Resonance Raman Spectroscopy, EPR Spectroscopy, and DFT Calculations for M-BIAIP

“…Our experimental results also reveal that the p K a value of phenolic OH of Cu II - BIAIP (9.8) is nearly the same as that of p -cresol (10.2). This similarity is compatible with the cross-linked tyrosine OH being protonated in the oxidized form when the pH is between 7.4 and 8.5 (Kandori et al), and in the oxidized and reduced form for a pH lower than 9 (Iwaki et al − ) based on FT-IR experimental results, in contrast with the proposal of Yoshikawa et al The conservation of the Cu II environment as well as phenolic OH protonation for Cu II - BIAIP for pH values between 5 and 10 is consistent with no change in the Cu B C−O frequency being observed for pH values between 5.5 and 9.7 for CO-bound cytochrome ba 3 from Thermus thermophilus , whereas the interpretation of the pH dependencies of Fe II -CO, Fe II C−O, and Cu I C−O is still controversial. − In other words, a role of proton delivery is unlikely for cross-linked phenol except when proton release is coupled with one-electron oxidation, because the p K a of the protonated phenoxyl radical form is remarkably low.…”

“…The upshift of 7a‘ (C−O stretching) by ca. 15 cm -1 is especially significant, suggesting that band assignments should be revised given the differential FT-IR spectra observed for enzymes. − , …”

ns-μs Time-Resolved Step-Scan FTIR of ba3 Oxidoreductase from Thermus thermophilus: Protonic Connectivity of w941-w946-w927

“…Finally, a peak/trough at 1506/1513 cm −1 is present and exhibits small intensity changes, but the ratio of the 1506/1513 cm −1 modes remained unchanged. This derivative form feature has been attributed to tyrosinate/tyrosine vibrations [31]. …”

Infrared Difference Spectroscopy of Proteins: From Bands to Bonds

“…As already mentioned (see section ), amide I vibrations are often coupled. As a consequence, 13 C labeling of one or few amide CO groups can affect the frequencies and intensities of several amide I bands. , Nevertheless, in exceptional cases, an amide I band in an IR difference spectrum can be assigned to a specific residue by amino acid-specific isotope labeling. This is possible when the natural amide I frequency of an amide CO group is high or low enough to be uncoupled from the rest of the amide CO groups in the protein.…”

“…Several labeling strategies have been used to assign vibrations of the phenol/phenolate group, in particular the ring vibration at 1520−1495 cm −1 and the coupled νC−O and δCOH vibrations at 1275−1150 cm −1 . Examples include the labeling of the carbon nearest to the hydroxyl group using [ring-4- 13 C]Tyr (see Figure 44), 601,632,633 the labeling of all the hydrogens in the phenolic ring using [ring-2 H 4 ]Tyr, 594,633,634 labeling all carbon atoms in the ring using [ring- 13 C 6 ]-Tyr, 289 or the labeling of the hydroxyl oxygen using 18 O-Tyr. 635 For His residues, the main vibrations of interest are the ring C−N vibrations of the imidazole group.…”