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The R481 residue in cytochrome c oxidase from Rhodobacter sphaeroides forms hydrogen bonds with the propionate groups of both heme a and heme a3. It has been postulated that R481 is the proton loading site in the proton exit pathway essential for proton translocation. A recent functional study showed that the mutations of R481 to His, Leu and Gln cause the reduction of the activity to ~5-18% of the native level, and the absence of proton pumping in R481Q but retention of ~40% efficiency in R481H and R481L (H.J. Lee, L. Öjemyr, A. Vakkasoglu, P. Brzezinski and R. B. Gennis, manuscript submitted). To decipher the molecular mechanism underlying the perturbed functionalities, we have used resonance Raman spectroscopy to examine the structural properties of the three mutants. The data show that the frequencies of the formyl C=O stretching modes of both the heme a and a3 in the mutants are characteristic of formyl groups exposed to an aqueous environment, indicating that the mutations disrupt the native H-bonding interaction between the formyl group of heme a and R52, as well as the hydrophobic environment surrounding the formyl group of heme a3. In addition to the change in the environments of heme a and a3, the Raman data show that the mutations induce a partial conversion of the heme a3 from a high-spin to a low-spin state, suggesting that the mutations are associated with the rearrangement of the CuB-heme a3 binuclear center. The Raman results reported here demonstrate that R481 plays a critical role in supporting efficient proton pumping, by holding the heme groups in a proper environment.
Cytochrome c oxidase (CcO) is the terminal enzyme of the electron transfer chain in mitochondria and many bacteria. It functions as a proton pump, by utilizing the chemical energy harvested from the four-electron reduction reaction of dioxygen to water. The enzyme is comprised of a binuclear center, containing the high-spin heme a3 and a nearby copper center, CuB, in which the chemistry takes place, and a low-spin heme a, through which the electrons pass from the substrate, cytochrome c, to the catalytic binuclear center . Crystallographic studies of bacterial CcO revealed two proton-conducting pathways, the D- and K-channels [2-4]. The importance of the two channels in supporting proton pumping have been demonstrated by a variety of site-directed mutagenesis studies [1, 5, 6]. Crystallographic studies of the bovine CcO, on the other hand, revealed a new H-channel [7, 8]. Although mutagenesis studies demonstrated that it is a functional proton pumping channel in the bovine enzyme , the H-channel does not seem to be operative in bacterial enzymes . Despite its importance, the molecular mechanism linking the structural/redox changes at the metal centers to the changes in the hydrogen bonding interactions in the proton pumping channels, which enables proton translocation in the protein matrix against the proton gradient, remains to be elucidated .
Considerable evidence indicates that in bacterial CcO, the D-channel translocates both chemical protons required for the oxygen chemistry and pumped protons. The E286 residue at the end of the D-channel has been proposed to act as a gate controlling the protons to be delivered to the binuclear center for chemistry or to the proton exit pathway for proton pumping. To drive proton pumping against the proton gradient, there must be a proton accepting site beyond E286, which undergoes changes in pKa in response to the oxygen chemistry occurring in the binuclear center . One such candidate is R481, which forms H-bonds with both heme a and a3. R481 is highly conserved in the heme-copper oxidase families of enzymes [11-15]. In the bo3 CcO from E. coli, the mutations of R481 to Gln or Leu decreased the activity to ~75 and 40%, respectively, although R481Q still pumped protons while R481L did not . In aa3 CcO from R. sphaeroides (RsCcO), the mutation of R481 to Lys, which retains a positive charge on the sidechain, does not affect the activity and proton pumping capability . Additional studies show that the mutation of R481 in RsCcO to His, Leu or Gln caused the activity to drop to ~18% (R481H) or 5% (R481L and R481Q) of the native level, and the proton pumping efficiency to decrease to ~40% (R481H and R481L) or 0% (R481Q) of the native level (H.J. Lee, L. Öjemyr, A. Vakkasoglu, P. Brzezinski and R. B. Gennis, manuscript submitted). The studies also revealed that the mutations alter the optical absorption spectra of the enzyme, decrease mid-point potentials of the two heme groups and affect the kinetics of the oxygen reaction, implying that the structural properties of the heme a and a3 are perturbed. These data confirm the importance of R481, but the molecular mechanism underlying the perturbed functionalities, especially the remarkable partial retention of the proton pumping efficiency of the H481L mutant (in which the Leu substituent is incapable of forming H-bond with the D propionate of heme a3) remains unclear.
In this work, we used resonance Raman spectroscopy to investigate how extensively the structural properties of rsCcO are disturbed by the R481H, R481L and R481Q mutations, and to address how the perturbed structural properties account for the perturbed functionality of the mutants.
R481H, R481L and R481Q were all constructed by using a Quik-Change mutagenesis kit from Stratagene. A PJS3-SH plasmid was used as a template for each mutation. The expression plasmids, pRK415-1, containing the mutations were first transferred into S-17-1 cells by electroporation, and the plasmids were then transferred into the Rhodobacter sphaeroides JS100 strain by conjugation. Sequencing was performed by the UIUC Biotech. Center. Cells were grown in Sistrom's minimum media (with 50 μg/mL streptomycin, 50 μg/mL spectinomycin, and 1μg/mL tetracycline) at 30 °C, until the growth reached the early stationary phase. Wild type and mutant enzymes were His-tagged and purified by histidine affinity chromatography.
The resonance Raman spectra were carried out as previously described . Briefly, the 413.1 nm excitation from a Kr ion laser (Spectra-Physics, Mountain View, CA) was focused to a ~30 μm spot on the spinning quartz cell rotating at ~1,000 rpm. The scattered light, collected at a right angle to the incident laser beam, was focused on the 100 μm - wide entrance slit of a 1.25 m Spex spectrometer equipped with a 1200 grooves/mm grating (& Lomb, Analytical Systems Division, Rochester, NY), where it was dispersed and then detected by a liquid nitrogen-cooled CCD detector (Princeton Instruments, Trenton, NJ). A holographic notch filter (Kaiser Optical Systems, Ann Arbor, MI) was used to remove the laser line. The Raman shift was calibrated with indene. The laser power was kept <4 mW for all measurements to avoid photo-damage to the protein.
As references for the R481H, L and Q mutants of rsCcO, the resonance Raman spectra of the wild type (wt) enzyme were measured. Figures 1a and and2a2a show the resonance Raman spectra of the wt enzyme in the fully oxidized and reduced states, respectively, in the high frequency region (~1300-1800 cm-1). The vibrational modes observed in this region of the spectra are sensitive to the electronic and structural properties of the hemes, such as the electron density in the porphyrin macrocycle and the spin- and coordination-states of the heme iron atoms, as well as the environment/orientation of the formyl group attached to the hemes. The assignments of these well-established vibrational modes, including the totally symmetric porphyrin skeletal vibrational modes [18, 19] and the formyl stretching modes (nC=O) , are indicated in Figures 1--2.2. The frequencies of the electron density marker line (n4) in the oxidized and reduced spectra at 1370 and 1357 cm-1, respectively, confirm the oxidation states of each of the samples.
As shown in Figure 1b-d, the oxidized forms of all the R481 mutants (R481H, R481L and R481Q) exhibit similar spectra. They comprise porphyrin skeletal vibrational modes akin to those of the wt protein, although the intensity ratios of the 1573 cm-1 band (the high-spin n 2,a3 mode) to the 1585 cm-1 band (the low-spin n 2a3 and n 2,a modes) are significantly reduced, as manifested in the difference spectra (e-g). The data indicate that the mutations induce a partial spin-conversion of heme a3 from high to low-spin, implying that in some population of each mutant of the heme a3 iron is coordinated by a strong field ligand. The degree of spin-conversion, unfortunately, can not be quantified as the spin sensitive lines partially overlap with other heme modes. In addition to the spin-state change, differences are apparent in the 1620-1690 cm-1 spectral region, containing the formyl vibrational modes of both heme a and a3. As shown by the difference spectra (e-g), the mutations introduce two new bands at ~1640 and ~1660 cm-1 at the expense of the formyl vibrational modes of hemes a and a3 at ~1646 and ~1671 cm-1, respectively, indicating the perturbation in the conformations of the formyl groups.
As the spectral assignments of the formyl modes in this spectral region are complicated by the presence of the n10 porphyrin skeletal vibrational mode at 1640 cm-1, which is associated with the low-spin component of heme a3 [20-22], we used curve-fitting method to analyze the difference spectra. As shown in the right panel of Figure 1, the R481H-wt difference spectrum can be fitted with two positive formyl modes centered at 1653 and 1662 cm-1, two negative formyl modes at 1649 and 1673 cm-1, and a n10 mode at 1640 cm-1. The data demonstrated that the mutations cause an upshift and downshift in the formyl vibrational frequencies of heme a and a3, respectively.
The reduced forms of all the mutants, like the oxidized forms, exhibit similar spectra (Figure 2b-d). As compared to the wt enzyme, the porphyrin skeletal vibrational modes are mostly unchanged, except that the relative intensity of the 1566 cm-1 band (the high-spin n 2,a3 mode) is slightly reduced, again indicating a partial conversion of the high-spin heme a3 to a low-spin species (which is most pronounced in the R481Q mutant) The difference spectra (e-g) in the 1560-1700 cm-1 region show that the mutations introduced new broad band(s) at ~1633 cm-1 at the expense of the formyl vibrational modes of hemes a and a3 at ~1610 and ~1662 cm-1, respectively. Curve-fitting of the R481H-wt difference spectrum show that the formyl vibrational modes of hemes a and a3 of the wt are at 1611 and 1664 cm-1, respectively; they shift to 1633 and 1636 cm-1, respectively, in the mutants. The data indicate that the formyl modes of heme a and heme a3 are upshifted and downshifted, respectively, in the mutants, as in the case of the oxidized mutants.
The resonance Raman data reported here clearly demonstrate that the mutation of R481 to H, L or Q in RsCcO significantly perturb the formyl vibrational modes of heme a and a3. Earlier Raman studies indicated that three major factors may modify the formyl vibrational mode of the a-type heme: (1) the redox state of the heme iron, (2) the electronic coupling between the formyl group and the heme macrocycle controlled by the relative orientation of the formyl group with respect to the heme plane, and (3) the hydrogen bonding interactions between the formyl group and its surrounding protein matrix [20, 23]. It is well-established that, in the ferrous form of the wt RsCcO, the heme a formyl group is strongly H-bonded with the surrounding protein matrix, as indicated by its low formyl nC=O frequency at 1610 cm-1 , which is consistent with the crystallographic data showing a strong H-bond between R52 and the formyl group (Figure 3). Upon oxidation, the mode shifts up by 36 cm-1, which is much larger than that predicted based on simply the change in the oxidation state of the heme iron (~25 cm-1 shift is anticipated based on the studies of an isolated heme a) . The data indicate that the oxidation of the heme iron weakens the H-bonding interaction between the formyl group of heme a and R52.
As shown in Figure 3 , the heme a, heme a3 and the CuB center are linked together via an extended H-bonding network mediated by R481, R482, the D-propionates of the two hemes, two water molecules and H334 (one of the CuB ligands). We infer that the mutations of R481 cause the movement of the heme a and a3, as they interrupt the linkage between the periphery of the two hemes, leaving only the two histidines (H419 and H421) from Helix-X to maintain their juxtaposition. The heme-movement interrupts the H-bonding interaction between the formyl group of heme a and R52, thereby accounting for the increase in the formyl nC=O frequency to values similar to that of the isolated heme a in an aqueous environment (Table 1). In contrast to heme a, the formyl group of heme a3 in the wt RsCcO is in a hydrophobic environment, as indicated by a very high formyl nC=O frequency in both oxidation states. Our Raman data indicate that the heme movement induced by the R481 mutations also causes the exposure of the formyl group of heme a3 to a new environment capable of donating stronger H-bond(s) to it, accounting for the downshift of the formyl nC=O frequency to that observed in an isolated heme a in an aqueous solvent. In addition to the movement of the heme a and a3, as reported by the formyl nC=O modes, the Raman data show that the mutations also induce partial conversion of the high-spin heme a3 to a low spin species. This suggests that the heme movement is associated with the rearrangement of the CuB-heme a3 center (due to the disruption of the H-bonding network involving H334), leading to the coordination of one of the histidine ligands of the CuB or an exogenous strong field ligand (such as a hydroxide) to the heme a3 ion.
In models derived from the studies of bacterial CcO [11-15], R481 has been postulated as the proton loading site in the proton exit channel that is essential for proton pumping. F unctional studies of the R481H, R481L and R481Q mutants show that the activity of the mutants are decreased to ~5-18% of the native level, in addition, the proton pumping is abolished in R481Q, but is retained at a ~40% level in both R481H and R481L (H.J. Lee, L. Öjemyr, A. Vakkasoglu, P. Brzezinski and R. B. Gennis, manuscript submitted). The Raman data demonstrate that the perturbed functionalities introduced by the mutations are a result of the disruption of the H-bonding network linking both hemes, as well as that connecting the heme groups to their peripheral environment, as illustrated in Figure 3. The data clearly demonstrate that R481 plays an important role in supporting efficient proton pumping, by holding the heme groups in an appropriate environment. They also show that R481 does not function as the proton loading site in the proton exit pathway, as its mutation to Leu, which is incapable of mediating proton transfer, does not abolish the proton pumping activity. It is conceivable that, by interrupting the extended H-bonding network mediated by R481 (Figure 3), the R481 mutations indirectly affect the functionality of the true proton loading site, such as the propionate group on ring A of heme a3 or one of the CuB histidine ligands, as we proposed in the past based on Raman studies of the aa3 oxidase from A. ambivalens .
On the basis of the studies of the mammalian CcO, Yoshikawa and coworkers proposed that the H-bonding interaction between the formyl group of heme a, located in the H channel, and R38 (equivalent to R52 in RsCcO) is essential for proton pumping [7, 9]. They hypothesize that the oxidation state of heme a alters the H-bonding interaction between R38 and the formyl group of the heme a, thereby mediating proton translocation. The observation that the R481L mutant of RsCcO still pump protons although the H-bonding interaction between R52 and the formyl group of heme a in this mutants is disrupted, demonstrates that the proton pumping model proposed by Yoshikawa and coworkers is not operative in RsCcO and supports the concept that the mammalian and bacterial enzymes follow distinct proton pumping mechanisms.
Taken together the structural information on the R481H, R481L, R481Q mutants of rsCcO revealed by our resonance Raman studies and the functional data reported by Lee et al. (H.J. Lee, L. Öjemyr, A. Vakkasoglu, P. Brzezinski and R. B. Gennis, manuscript submitted) call for a re-evaluation of the nature of the proton loading site that is critical for the proton translocation in CcO.
This work was supported by the National Institute of Health Grants GM074982 to D. L. R. and HL016101 to R. B. G.
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