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

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 Cϵ–Nϵ covalent bond with one of the CuB ligand histidines and has been proposed to be an acid-base catalyst essential for the O–O bond cleavage at the Oxy-to-P transition of the dioxygen reduction cycle (Uchida, T., Mogi, T., and Kitagawa, T. (2000) Biochemistry 39, 6669–6678). To probe structural changes at tyrosine residues, we examined redox difference Fourier transform infrared difference spectra of the wild-type enzyme in which either l-[1-13C]Tyr or l-[4-13C]Tyr has been biosynthetically incorporated in the tyrosine auxotroph. Spectral comparison between [1-13C]Tyr-labeled and unlabeled proteins indicated that substitution of the main chain carbonyl of a Tyr residue(s) significantly affected changes in the amide-I (∼1620–1680 cm–1) and -II (∼1540–1560 cm–1) regions. In contrast, spectral comparison between [4-13C]Tyr-labeled and unlabeled proteins showed only negligible changes, which was the case for both the pulsed and the resting forms. Thus, protonation of an OH group of tyrosines including Tyr-288 in the vicinity of the heme o-CuB binuclear center was not detected at pH 7.4 upon full reduction of cytochrome bo. Redox-induced main chain changes at a Tyr residue(s) are associated with structural changes at Glu-286 near the binuclear metal centers and may be related to switching of the K-channel operative at the reductive phase to D-channel at the oxidative phase of the dioxygen reduction cycle via conformational changes in the middle of helix VI.

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)(2)(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)(13)(14)(15)(16)) and x-ray crystallographic (17)(18)(19)(20)(21)(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). However, calculations of electrostatic energy on the P. denitrificans cytochrome c oxidase indicate that Tyr-288 is protonated in the oxidized state when a negative ligand such as OH Ϫ is bound in the binuclear center (34).
Time-resolved spectroscopic studies on bovine cytochrome c oxidase have identified a number of intermediates in the reaction of dioxygen reduction (see Refs. 35 (37)(38)(39)(40)(41)(42) and by radioactive iodide labeling followed by peptide mapping (31). Subsequent one-electron transfer to the binuclear center reduces the tyrosine neutral radical and converts the P species to the ferryl (F) species (Fe o 4ϩ ϭO, Cu B 2ϩ , Tyr-O Ϫ ). The F intermediate is converted to the oxidized form by a further electron transfer.
Crystallographic (19 -21) and protein sequencing (43) studies on cytochrome c oxidase revealed the presence of a peculiar C ⑀ -N ⑀ covalent bond between Tyr-288 and His-284, one of three histidine ligands of Cu B . The OH group of Tyr-288 could form a hydrogen bond with a diatomic ligand like dioxygen, favoring the cross-linked tyrosine to participate in dioxygen reduction. Studies with model compounds showed that the His-Tyr linkage lowers a pK a of the phenol moiety by 1.1 to 1. Fourier transform infrared (FTIR) 3 spectroscopy is a powerful technique to probe the molecular environment of the active center of terminal oxidases. Application of spectro-electrochemical cells (47)(48)(49)(50)(51)(52), attenuated total reflection FTIR apparatus (41,42,(53)(54)(55)(56), and a photochemical reduction technique (57-60) enabled us to examine protein structural changes upon reduction of the redox metal centers of respiratory terminal oxidases. Alternatively, photodissociation of CO from the reduced enzyme revealed the local structural change surrounding the heme-copper binuclear center of the E. coli cytochrome bo (61,62). Previous studies using site-directed mutants revealed that a COOH group of Glu-286 at the end of the D-channel undergoes hydrogen bond changes upon full reduction of the metal centers (48,50,59,60) or CO photolysis of the reduced, CO-bound enzyme (61,62). In contrast, a lack of Cu B and the substitution of high spin heme o for heme b in the Tyr-288 mutants (63,64) obscured protein structural changes attributable to Tyr-288 at the end of the K-channel (59). Recently, it has been reported that Glu-286 deprotonates upon one-electron reduction of the fully oxidized enzyme (42) or reverse electron transfer in the mixed valence enzyme (65).
In the present study, to probe redox-induced structural changes at tyrosines, we prepared the wild-type cytochrome bo into which either L-[1-13 C]Tyr or L-[4-13 C]Tyr (Fig. 1B) had been biosynthetically incorporated and measured their photo-reduced minus oxidized redox difference FTIR spectra. We found that redox-induced main chain changes at a Tyr residue(s) are associated with structural changes at Glu-286 near the binuclear metal center. In addition, protonation of an OH group of tyrosines was not detected at pH 7.4 and 8.5 under steadystate conditions. On the basis of present findings, we will discuss redoxinduced protein structural changes in subunit I.
Preparation of the Pulsed Form-A pulsed form of cytochrome bo was prepared according to Moody and Rich (69) as follows. The resting enzyme (ϳ0.25 mM) in 50 mM Tricine-NaOH (pH 8.5) containing 0.1% sucrose monolaurate and 0.5 mM sodium EDTA were incubated aerobically in the dark with 8 mM sodium ascorbate (pH 7.6) and 0.1 mM phenazine methosulfate (final concentrations) at 23°C for 1 h. Then the sample was dialyzed at 4°C against 300 volumes of 50 mM Tricine-NaOH (pH 8.5) containing 0.1% sucrose monolaurate and 0.5 mM sodium EDTA with four changes of the buffer every hour. After the dialysis, insoluble materials were removed by centrifugation at 16,000 rpm (22,640 ϫ g) and 4°C for 10 min. The sample solution was concentrated to a final concentration of about 0.5 mM using a Centricon 100 apparatus (Amicon) for 2 h at 4°C. The control sample (the resting form) was prepared as for the pulsed form except for the omission of the reduction with 8 mM sodium ascorbate and 0.1 mM phenazine methosulfate. The pulsed form showed the Soret peak at 407 nm and an intense g ϭ 3.7 signal, whereas the resting form had its Soret peak at 409 nm, consistent with previous reports (69).
FTIR Spectroscopy-The samples for spectroscopic analysis were prepared essentially according to Lübben and Gerwert (57). Five l of the reaction mixture containing about 0.25 mM air-oxidized enzyme (resting form), 0.25 mM riboflavin, 50 mM sodium EDTA, 50 mM sodium phosphate (pH 7.4), and 0.1% sucrose monolaurate was placed on a BaF 2 window and concentrated to some extent in a vacuum desiccator (59). The hydrated enzyme/detergent paste was then sandwiched by another BaF 2 window and covered by an aperture of 6-mm diameter. Once the enzyme was mixed with riboflavin, all of the procedures were carried out under dim red light conditions.
Oxford cryostat (DN-1704) was equipped in the FTIR spectrometer, and the sample was attached to the sample holder for the cryostat (70). Photoreduction of the air-oxidized enzymes was conducted at 15°C in the presence of riboflavin as a photoactivatable electron donor (63). The 256 interferograms at 2 cm Ϫ1 resolution were recorded with a Bio-Rad FTS-40 spectrometer and converted to the infrared absorption spectrum by use of a reference interferogram recorded in the absence of the sample. The spectral difference before and after irradiation was compared with the base line as the difference between the two spectra without intervening irradiation, and if necessary the base line value was subtracted from the data. The spectra in Figs. 2 and 3 are the averages of 4 -6 independent measurements.
In bovine cytochrome c oxidase, two negative bands at 1748 -1749 and 1737-1738 cm Ϫ1 were identified in the fully reduced minus oxidized difference spectrum in the unligated enzyme (49, 52, 54 -56, 71). As postulated by crystallographic studies (20), the latter signal has been tentatively assigned to the deprotonation of Asp-51 in subunit I (49,71). However, the 1588 cm Ϫ1 band assigned to a asym (COO Ϫ ) mode from Asp-51 (49) can be found in cytochrome c oxidase from Rhodobacter sphaeroides (52), in which the mammalian-specific Asp-51 is not conserved. Similar spectral changes in the oxidized cyanide-bound (53) and mixed valence CO-bound (71) enzymes indicate that deprotonation of carboxylic acid residues is associated with the reduction of Cu A /heme a. Recently, McMahon et al. (65) showed that reverse electron transfer in the mixed valence enzyme (i.e. reduction of heme a) induces the deprotonation of Glu-286 with a pK a of ϳ8.3 ((Ϫ)1735/(ϩ)1412 ( sym -(COO Ϫ )) cm Ϫ1 in D 2 O). In cytochrome c oxidase from R. sphaeroides, Nyquist et al. (42) reported that Glu-286 undergoes the deprotonation upon one-electron reduction (E state) and then the reprotonation upon two-electron reduction (R 2 state).
In contrast, a symmetrical feature of the (COOH) mode in the fully reduced minus oxidized difference spectra has been reported for cytochrome bo ((Ϫ)1745/(ϩ)1735 cm Ϫ1 (58)) and cytochrome c oxidase from P. denitrificans ((Ϫ)1746/(ϩ)1732 cm Ϫ1 (49, 54)) and R. sphaeroides ((Ϫ)1745/(ϩ)1735 cm Ϫ1 (56)). The H/D exchange affected both signals in cytochrome c oxidases (49, 56) but only the negative signal in cytochrome bo (58). Prutsch et al. (60) examined the photo-reduced minus mixed valence CO-bound difference spectrum of cytochrome bo and found a similar spectral change. These data are consistent with a change in the hydrogen-bonding environment of the protonated carboxylic side chain of Glu-286 rather than deprotonation upon reduction of low spin heme b (or heme a in cytochrome c oxidase).
Spectral features in the redox difference spectrum of [1-13 C]Tyr-WT (Fig. 2c) are considerably different from those of 12 C-WT and [4-13 C]Tyr-WT (Fig. 2, a and b), particularly in the amide-I region (1620 -1680 cm Ϫ1 ). This indicates that peptide carbonyl groups of tyrosine residues undergo structural changes upon full reduction of the redox centers in subunit I. In contrast, the similarity in the spectra between 12 C-WT and [4-13 C]Tyr-WT suggests that changes in protonation and/or environment do not occur for the OH group of tyrosines between the fully photo-reduced and the air-oxidized, resting forms.
Redox-induced Infrared Spectral Changes of Oxygen-pulsed Form at pH 8.5-It is known that the structure of the heme-Cu B binuclear center of the resting oxidized form (as prepared form) is different from that of the fully oxidized form in the catalytic cycle (i.e. oxygen-pulsed form) (3,69). It is possible that the protonation state of tyrosine side chains such as Tyr-288 is different between the two fully oxidized states. Therefore, we then compared the photo-induced redox difference spectra of the resting and pulsed forms at pH 8.5, at which the latter can be stably maintained.
Photo-reduced minus oxidized infrared difference spectra from the resting form at pH 7.4 and 8.5 and the pulsed form at pH 8.5 were very similar (not shown). This indicates that there are no pH effects between 7.4 and 8.5 in the resting form and no significant difference in structural changes between the resting and pulsed forms. Below, we examined the structural changes of side chain and peptide backbone of tyrosine residues in detail.
Redox-induced Structural Changes of Tyrosine Side Chain in Cytochrome bo-Tyrosine side chain has characteristic vibrations (72,73). Among them, a band in the 1200 -1300 cm Ϫ1 region is highly sensitive to the 13 C label at position 4 and has been assigned to the C-O stretching mode ( 7Јa ) that also includes the CO-H bending mode (␦(COH)). In fact, frequencies of tyrosine and tyrosinate (1250 and 1270 cm Ϫ1 , respectively) in aqueous solution exhibit a downshift to 1228 and 1246 cm Ϫ1 , respectively, for [4-13 C]Tyr (74). Such a downshift by 20 -30 cm Ϫ1 is also reported in proteins, such as photosystem II core complex (74 -76) and bacteriorhodopsin (77). Recently, FTIR studies on synthetic models for the His-Tyr cross-link and oxidases demonstrated that a prominent band around 1540 cm Ϫ1 is assignable to the protonated form of the cross-linked tyrosine (42,51,55,78).
The spectral comparison of photo-reduced minus air-oxidized forms for [4-13 C]Tyr-labeled and unlabeled cytochrome bo in the 1180 -1330 cm Ϫ1 region and at around 1540 cm Ϫ1 showed no isotope-sensitive spectral changes in the typical frequency region of tyrosine side chains and for the cross-linked tyrosine (not shown). This suggests that changes in protonation and/or the environment of tyrosine side chains are not involved in the reduction process of the resting oxidized form.  SEPTEMBER 23, 2005 • VOLUME 280 • NUMBER 38

JOURNAL OF BIOLOGICAL CHEMISTRY 32823
We found also that such changes are not involved in the reduction process of the pulsed-oxidized form (not shown).
Redox-induced Structural Changes of Peptide Backbone of Tyrosine Residues in Cytochrome bo-In contrast to the results of the isotope label of the tyrosine side chain ([4-13 C]Tyr-WT) the isotope label of the peptide carbonyl of tyrosine residues ([1-13 C]Tyr-WT) yields significant changes in the redox-induced difference spectra (Fig. 2). In Fig. 3a, photo-reduced minus resting oxidized infrared spectra of [4-13 C]Tyr-WT (solid line) and unlabeled 12 C-WT (dotted line) exhibit similar spectra, except that the base line of the former is lower than that of the latter in the 1700 -1600 cm Ϫ1 region. In particular, the amplitude of amide-I vibration in the 1670 -1620 cm Ϫ1 is identical between them. Results indicate that vibrational modes of the tyrosine side chain are not involved in this region and in the 1300 -1200 cm Ϫ1 region. Fig. 3b compares photo-reduced minus resting oxidized infrared spectra of [1-13 C]Tyr-WT (solid line) and unlabeled 12 C-WT (dotted line). In [1-13 C]Tyr-WT, a sharp positive peak at 1663 cm Ϫ1 significantly decreases its intensity, and two negative peaks at 1657 and 1647 cm Ϫ1 also decrease their intensities. On the other hand, a peak pair newly appears at (Ϫ)1612/(ϩ)1604 cm Ϫ1 . Because the 13 C isotope shift of amide-I vibration is expected to be 35-40 cm Ϫ1 , the peak pair is most likely to be shifted from the peak at (Ϫ)1647/(ϩ)1636 cm Ϫ1 . The peak at (ϩ)1663/(Ϫ)1657 cm Ϫ1 would shift to the 1630 -1620 cm Ϫ1 region, though the 13 CϭO stretching vibrations are not clearly observed. Thus, all prominent amide-I vibrations ((ϩ)1663, (Ϫ)1657, (Ϫ)1647, and (ϩ)1636 cm Ϫ1 ) (Fig. 3) in the photo-reduced minus resting oxidized difference spectrum involve that of tyrosine residue. A decrease of the amplitude by about one-half in [1-13 C]Tyr-labeled cytochrome bo suggests that peptide carbonyls of tyrosine residues are actively involved in the redox-induced protein structural changes.

Redox-induced Protein Structural Changes in Terminal
Oxidases-X-ray crystallographic studies on the oxidized and reduced forms of cytochrome c oxidase (2.30 and 2.35 Å, respectively, in bovine (20) and 3.0 and 3.3 Å, respectively, in P. denitrificans (79)) did not find any significant protein structural changes around the redox metal centers in subunit I. FTIR studies on bacterial (42, 48, 58 -60) and bovine (49, 52, 54 -56, 71) oxidases revealed the redox-induced protonation or hydrogen-bonding changes at the COOH group of Glu-286, which is located at the end of the D-channel (Fig. 1A). In cytochrome bo, hydrogen bonding changes in two Cys-SH groups have been identified also (59). One-electron reduction of the oxidized form (42), reverse electron transfer in the mixed valence CO-bound form (65), and reduction of the mixed valence CO-bound (60,71) form indicate that Glu-286 senses the redox state of low spin heme b (or heme a in cytochrome c oxidase). Such structural changes may be related to the switching of the K-channel operative at the initial reductive phase of dioxygen reduction to D-channel (12,59). It should be noted that Iwaki et al. (80) recently identified redox-linked histidine protonation changes in iron-sulfur protein of the cytochrome bc 1 complex.
Protonation State of Cross-linked Tyr-288 in Oxidized Form-Tyr-288 is located at the end of the K-channel (17)(18)(19)(20)(21)(22) and delivers one (or two) chemical protons to the heme-Cu B binuclear center (12,(25)(26)(27)(28). The presence of a peculiar C ⑀ -N ⑀ covalent bond between Tyr-288 and His-284 (19 -21, 43), one of three histidine ligands of Cu B , lowers the pK a of the phenol moiety (44 -46). The OH group of Tyr-288 could form a hydrogen bond with a diatomic ligand like dioxygen (20), favoring the cross-linked tyrosine to participate in dioxygen reduction (25, 26, 29 -32). Yoshikawa et al. (20) proposed that the cross-linked tyrosine is deprotonated in the fully oxidized state and upon reduction of the binuclear center Tyr-288-O Ϫ would serve as a proton acceptor for neutralization of the increased negative charge at the binuclear center (26). The present FTIR study on the [4-13 C]Tyr-labeled cytochrome bo indicates the absence of the redox-induced protonation changes at tyrosine residues ( Figs. 2 and 3). Hellwig et al. (51) examined electrochemically induced FTIR difference spectra of the D 4 Tyr-labled cytochrome c oxidase from P. denitrificans, and found no clear evidence for the protonation change of Tyr-288. Electrostatic calculations on the P. denitrificans oxidase suggest that tyrosines in the oxidase are protonated at neutral pH if OH Ϫ is present in the heme-copper binuclear center (34). In the F-to-O transition of cytochrome c oxidase, Nyquist et al. (42) identified the C-O stretching mode of tyrosine at (Ϫ)1248 cm Ϫ1 , the 19 phenyl ring stretching mode at (Ϫ)1515/(ϩ)1499 cm Ϫ1 (tyrosine and tyrosinate, respectively), and the (Ϫ)1546 cm Ϫ1 band assignable to a coupled His-Tyr ring mode with large contributions from the C-N of the covalent bond between both ring systems (78). Three negative bands at 1455, 1515, and 1546 cm Ϫ1 were also found in a His-Tyr cross-linked model (2-(4-methyl-1H-imidazol-1-yl)-4-methylphenol)) (78). These results suggest that the cross-linked Tyr-288 is protonated in the oxidized state.  ϳ1519 cm Ϫ1 in the P M minus O difference spectrum (41). The 1519cm Ϫ1 band could be assigned to a C-O stretching vibration of the tyrosine radical (1516 cm Ϫ1 in the model compound) (46). In cytochrome c oxidase from R. sphaeroides, bands at 1517 and 1587 cm Ϫ1 are associated with the P M intermediate (42). The presence of the (Ϫ)1515-cm Ϫ1 band (tyrosine) in the F minus O difference spectrum (42) indicates the reduction of the tyrosine radical to tyrosinate in the P-to-F transition and the reprotonation in the F-to-O transition. These findings suggest that the cross-linked Tyr donates hydrogen to the bound dioxygen to facilitate the O-O bond scission during the P formation and reprotonates at the oxidized state after receiving one electron at the F state.

Role of Cross-linked Tyr-288 in Dioxygen Reduction
In conclusion, we examined the photo-reduced minus oxidized FTIR difference spectra of the wild-type cytochrome bo, where either L-[1-13 C] or L-[4-13 C]Tyr has been biosynthetically incorporated, and found that redox-linked structural changes at a tyrosine residue(s) are associated with hydrogen bonding changes at Glu-286 in subunit I. Spectral comparison between [4-13 C]Tyr-labeled and unlabeled proteins indicates that Tyr-288 remains protonated upon reduction of the enzyme to serve as a hydrogen donor to the bound dioxygen at the P formation.