FTIR studies of internal proton transfer reactions linked to inter-heme electron transfer in bovine cytochrome c oxidase

FTIR difference spectroscopy is used to reveal changes in the internal structure and amino acid protonation states of bovine cytochrome c oxidase (CcO) that occur upon photolysis of the CO adduct of the two-electron reduced (mixed valence, MV) and four-electron reduced (fully reduced, FR) forms of the enzyme. FTIR difference spectra were obtained in D(2)O (pH 6-9.3) between the MV-CO adduct (heme a(3) and Cu(B) reduced; heme a and Cu(A) oxidized) and a photostationary state in which the MV-CO enzyme is photodissociated under constant illumination. In the photostationary state, part of the enzyme population has heme a(3) oxidized and heme a reduced. In MV-CO, the frequency of the stretch mode of CO bound to ferrous heme a(3) decreases from 1965.3 cm(-1) at pH* 相似文献   

Demonstration by FTIR that the bo-type ubiquinol oxidase of Escherichia coli contains a heme-copper binuclear center similar to that in cytochrome c oxidase and that proper assembly of the binuclear center requires the cyoE gene product.

Amino acid sequence data have revealed that the bo-type ubiquinol oxidase from Escherichia coli is closely related to the eukaryotic aa3-type cytochrome c oxidases. In the cytochrome c oxidases, the reduction of oxygen to water occurs at a binuclear center comprised of heme a3 and Cu(B). In this paper, Fourier transform infrared (FTIR) spectroscopy of CO bound to the enzyme is used to directly demonstrate that the E. coli bo-type ubiquinol oxidase also contains a heme-copper binuclear center. Photolysis of CO ligated to heme o at low temperatures (e.g., 30 K) results in formation of a CO-Cu complex, showing that there is a heme-Cu(B) binuclear center similar to that formed by heme a3 and Cu(B) in the eukaryotic oxidase. It is further demonstrated that the cyoE gene product is required for the correct assembly of this binuclear center, although this polypeptide is not required as a component of the active enzyme in vitro. The cyoE gene product is homologous to COX10, a nuclear gene product from Saccharomyces cerevisiae, which is required for the assembly of yeast cytochrome c oxidase. Deletion of the cyoE gene results in an inactive quinol oxidase that is, however, assembled in the membrane. FTIR analysis of bound CO shows that Cu(B) is present in this mutant but that the heme-Cu(B) binuclear center is abnormal. Analysis of the heme content of the membrane suggests that the cyoE deletion results in the insertion of heme B (protoheme IX) in the binuclear center, rather than heme O.(ABSTRACT TRUNCATED AT 250 WORDS)     

Proton pumping and oxidase activity of thermophilic cytochrome oxidase remain after its extensive proteolysis

A proton-pumping heme aa3-type cytochrome oxidase purified from the thermophilic bacterium PS3 was treated with trypsin, thermolysin, chymotrypsin, subtilisin, or pronase. The cleavage of the oxidase subunits and the effects of their cleavage on the oxidase activity and proton-pumping in reconstituted vesicles were studied. Trypsin and thermolysin cleaved some of the oxidase subunits without affecting the proton-pumping, but subtilisin and pronase cleaved all the subunits resulting in partial decrease in both activities. Chymotrypsin had an intermediate effect. Subunit II of this enzyme contains heme c which is also cleaved by proteases.     

Differential effects of glutamate-286 mutations in the aa(3)-type cytochrome c oxidase from Rhodobacter sphaeroides and the cytochrome bo(3) ubiquinol oxidase from Escherichia coli

Both the aa(3)-type cytochrome c oxidase from Rhodobacter sphaeroides (RsCcO(aa3)) and the closely related bo(3)-type ubiquinol oxidase from Escherichia coli (EcQO(bo3)) possess a proton-conducting D-channel that terminates at a glutamic acid, E286, which is critical for controlling proton transfer to the active site for oxygen chemistry and to a proton loading site for proton pumping. E286 mutations in each enzyme block proton flux and, therefore, inhibit oxidase function. In the current work, resonance Raman spectroscopy was used to show that the E286A and E286C mutations in RsCcO(aa3) result in long range conformational changes that influence the protein interactions with both heme a and heme a(3). Therefore, the severe reduction of the steady-state activity of the E286 mutants in RsCcO(aa3) to ~0.05% is not simply a result of the direct blockage of the D-channel, but it is also a consequence of the conformational changes induced by the mutations to heme a and to the heme a(3)-Cu(B) active site. In contrast, the E286C mutation of EcQO(bo3) exhibits no evidence of conformational changes at the two heme sites, indicating that its reduced activity (3%) is exclusively a result of the inhibition of proton transfer from the D-channel. We propose that in RsCcO(aa3), the E286 mutations severely perturb the active site through a close interaction with F282, which lies between E286 and the heme-copper active site. The local structure around E286 in EcQO(bo3) is different, providing a rationale for the very different effects of E286 mutations in the two enzymes. This article is part of a Special Issue entitled: Allosteric cooperativity in respiratory proteins.     

Photothermal studies of CO photodissociation from mixed valence Escherichia coli cytochrome bo3

Here, we report the volume and enthalpy changes accompanying CO photodissociation from the mixed valence form of cytochrome bo3 oxidase from Escherichia coli. The results of photoacoustic calorimetry indicate two kinetic phases with distinct volume and enthalpy changes accompanying CO photodissociation from heme o3 and its transfer to CuB. The first phase occurring on a timescale of <50 ns is characterized by a volume decrease of -1.3+/-0.3 mL mol-1 and enthalpy change of 32+/-1.6 kcal mol-1. Subsequently, a volume increase of 2.9 mL mol-1 with an enthalpy change of -5.3+/-2.5 kcal mol-1 is observed with the lifetime of approximately 250 ns (this phase has not been detected in previous optical studies). These volume and enthalpy changes differ from the volume and enthalpy changes observed for CO dissociation from fully reduced cytochrome bo3 oxidase indicating that the heme o3/CuB active site dynamics are affected by the redox state of heme b.     

Ligand binding to heme proteins: III. FTIR studies of His-E7 and Val-E11 mutants of carbonmonoxymyoglobin.

Fouier-transform infrared (FTIR) difference spectra of several His-E7 and Val-E11 mutants of sperm whale carbonmonoxymyoglobin were obtained by photodissociation at cryogenic temperatures. The IR absorption of the CO ligand shows characteristic features for each of the mutants, both in the ligand-bound (A) state and in the photodissociated (B) state. For most of the mutants, a single A substate band is observed, which points to the crucial role of the His-E7 residue in determining the A substrate spectrum of the bound CO in the native structure. The fact that some of the mutants show more than one stretch band of the bound CO indicates that the appearance of multiple A substates is not exclusively connected to the presence of His-E7. In all but one mutant, multiple stretch bands of the CO in the photodissociated state are observed; these B substates are thought to arise from discrete positions and/or orientations of the photodissociated ligand in the heme pocket. The red shifts of the B bands with respect to the free-gas frequency indicate weak binding in the heme pocket. The observation of similar red shifts in microperoxidase (MP-8), where there is no residue on the distal side, suggests that the photodissociated ligand is still associated with the heme iron. Photoselection experiments were performed to determine the orientation of the bound ligand with respect to the heme normal by photolyzing small fractions of the sample with linearly polarized light at 540 nm.(ABSTRACT TRUNCATED AT 250 WORDS)     

Fourier transform infrared (FTIR) and step-scan time-resolved FTIR spectroscopies reveal a unique active site in cytochrome caa3 oxidase from Thermus thermophilus

Fourier transform infrared (FTIR) and step-scan time-resolved FTIR difference spectra are reported for the [carbonmonoxy]cytochrome caa(3) from Thermus thermophilus. A major C-O mode of heme a(3) at 1958 cm(-1) and two minor modes at 1967 and 1975 cm(-1) (7:1:1) have been identified at room temperature and remained unchanged in H(2)O/D(2)O exchange. The observed C-O frequencies are 10 cm(-1) higher than those obtained previously at 21 K (Einarsdóttir, O., Killough, P. M., Fee, J. A., and Woodruff, W. H. (1989) J. Biol. Chem. 264, 2405-2408). The time-resolved FTIR data indicate that the transient Cu(B)(1+)-CO complex is formed at room temperature as revealed by the CO stretching mode at 2062 cm(-1). Therefore, the caa(3) enzyme is the only documented member of the heme-copper superfamily whose binuclear center consists of an a(3)-type heme of a beta-form and a Cu(B) atom of an alpha-form. These results illustrate that the properties of the binuclear center in other oxidases resulting in the alpha-form are not required for enzymatic activity. Dissociation of the transient Cu(B)(1+)-CO complex is biphasic. The rate of decay is 2.3 x 10(4) s(-1) (fast phase, 35%) and 36.3 s(-1) (slow phase, 65%). The observed rate of rebinding to heme a(3) is 34.1 s(-1). The implications of these results with respect to the molecular motions that are general to the photodynamics of the binuclear center in heme-copper oxidases are discussed.     

Probing protonation/deprotonation of tyrosine residues in cytochrome ba3 oxidase from Thermus thermophilus by time-resolved step-scan Fourier transform infrared spectroscopy

Elucidating the properties of the heme Fe-Cu(B) binuclear center and the dynamics of the protein response in cytochrome c oxidase is crucial to understanding not only the dioxygen activation and bond cleavage by the enzyme but also the events related to the release of the produced water molecules. The time-resolved step-scan FTIR difference spectra show the ν(7a)(CO) of the protonated form of Tyr residues at 1247 cm(-1) and that of the deprotonated form at 1301 cm(-1). By monitoring the intensity changes of the 1247 and 1301 cm(-1) modes as a function of pH, we measured a pK(a) of 7.8 for the observed tyrosine. The FTIR spectral changes associated with the tyrosine do not belong to Tyr-237 but are attributed to the highly conserved in heme-copper oxidases Tyr-136 and/or Tyr-133 residue (Koutsoupakis, K., Stavrakis, S., Pinakoulaki, E., Soulimane, T., and Varotsis, C. (2002) J. Biol. Chem. 277, 32860-32866). The oxygenation of CO by the mixed-valence form of the enzyme revealed the formation of the ～607 nm P (Fe(IV)=O) species in the pH 6-9 range and the return to the oxidized form without the formation of the 580 nm F form. The data indicate that Tyr-237 is not involved in the proton transfer pathway in the oxygenation of CO by the mixed-valence form of the enzyme. The implication of these results with respect to the role of Tyr-136 and Tyr-133 in proton transfer/gating along with heme a(3) ring D propionate-H(2)O-ring A propionate-Asp-372 site to the exit/output proton channel (H(2)O pool) is discussed.     

Observation of the equilibrium CuB-CO complex and functional implications of the transient heme a3 propionates in cytochrome ba3-CO from Thermus thermophilus. Fourier transform infrared (FTIR) and time-resolved step-scan FTIR studies

We report the first evidence for the existence of the equilibrium Cu(B)1+-CO species of CO-bound reduced cytochrome ba(3) from Thermus thermophilus at room temperature. The frequency of the C-O stretching mode of Cu(B)1+-CO is located at 2053 cm(-1) and remains unchanged in H(2)O/D(2)O exchanges and, between pD 5.5 and 9.7, indicating that the chemical environment does not alter the protonation state of the Cu(B) histidine ligands. The data and conclusions reported here are in contrast to the changes in protonation state of Cu(B)-His-290, reported recently (Das, T. K., Tomson, F. K., Gennis, R. B., Gordon, M., and Rousseau, D. L. (2001) Biophys. J. 80, 2039-2045 and Das, T. P., Gomes, C. M., Teixeira, M., and Rousseau, D. L. (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 9591-9596). The time-resolved step-scan FTIR difference spectra indicate that the rate of decay of the transient Cu(B)1+-CO complex is 34.5 s(-1) and rebinding to heme a(3) occurs with k(2) = 28.6 s(-1). The rate of decay of the transient Cu(B)1+-CO complex displays a similar time constant as the absorption changes at 1694(+)/1706(-), attributed to perturbation of the heme a(3) propionates (COOH). The nu(C-O) of the transient Cu(B)1+-CO species is the same as that of the equilibrium Cu(B)1+-CO species and remains unchanged in the pD range 5.5-9.7 indicating that no structural change takes place at Cu(B) between these states. The implications of these results with respect to proton pathways in heme-copper oxidases are discussed.     

Activation volumes for intramolecular electron transfer in Escherichia coli cytochrome bo(3)

In this report we describe the activation volumes associated with the heme-heme electron transfer (ET) and CO rebinding to the binuclear center subsequent to photolysis of the CO-mixed-valence derivative of Escherichia coli cytochrome bo(3) (Cbo). The activation volumes associated with the heme-heme ET (k=1.2 x 10(5) s(-1)), and CO rebinding (k=57 s(-1)) are found to be +27.4 ml/mol and -2.6 ml/mol, respectively. The activation volume associated with the rebinding of CO is consistent with previous Cu X-ray absorption studies of Cbo where a structural change was observed at the Cu(B) site (loss of a histidine ligand) due to a change in the redox state of the binuclear center. In addition, the volume of activation for the heme-heme ET was found to be quite distinct from the activation volumes obtained for heme-heme ET in bovine heart Cytochrome c oxidase. Differences in mechanisms/pathways for heme b/heme o(3) and heme a/heme a(3) ET are suggested based on the associated activation volumes and previously obtained Marcus parameters.     

pH-dependent structural changes at the Heme-Copper binuclear center of cytochrome c oxidase

The resonance Raman spectra of the aa3 cytochrome c oxidase from Rhodobacter sphaeroides reveal pH-dependent structural changes in the binuclear site at room temperature. The binuclear site, which is the catalytic center of the enzyme, possesses two conformations at neutral pH, assessed from their distinctly different Fe-CO stretching modes in the resonance Raman spectra of the CO complex of the fully reduced enzyme. The two conformations (alpha and beta) interconvert reversibly in the pH 6-9 range with a pKa of 7.4, consistent with Fourier transform infrared spectroscopy measurements done at cryogenic temperatures (D.M. Mitchell, J.P. Sapleigh, A.M.Archer, J.O. Alben, and R.B.Gennis, 1996, Biochemistry 35:9446-9450). It is postulated that the different structures result from a change in the position of the Cu(B) atom with respect to the CO due to the presence of one or more ionizable groups in the vicinity of the binuclear center. The conserved tyrosine residue (Tyr-288 in R. sphaeroides, Tyr-244 in the bovine enzyme) that is adjacent to the oxygen-binding pocket or one of the histidines that coordinate Cu(B) are possible candidates. The existence of an equilibrium between the two conformers at physiological pH and room temperature suggests that the conformers may be functionally involved in enzymatic activity.     

Time-resolved step-scan Fourier transform infrared investigation of heme-copper oxidases: implications for O2 input and H2O/H+ output channels

We have applied FTIR and time-resolved step-scan Fourier transform infrared (TRS(2)-FTIR) spectroscopy to investigate the dynamics of the heme-Cu(B) binuclear center and the protein dynamics of mammalian aa(3), Pseudomonas stutzeri cbb(3), and caa(3) and ba(3) from Thermus thermophilus cytochrome oxidases. The implications of these results with respect to (1) the molecular motions that are general to the photodynamics of the binuclear center in heme-copper oxidases, and (2) the proton pathways located in the ring A propionate of heme a(3)-Asp372-H(2)O site that is conserved among all structurally known oxidases are discussed.     

The role of the cross-link His-Tyr in the functional properties of the binuclear center in cytochrome c oxidase

Resonance Raman and Fourier transform infrared spectroscopies have been used to study the aa(3)-type cytochrome c oxidase and the Y280H mutant from Paracoccus denitrificans. The stability of the binuclear center in the absence of the Tyr(280)-His(276) cross-link is not compromised since heme a(3) retains the same proximal environment, spin, and coordination state as in the wild type enzyme in both the oxidized and reduced states. We observe two C-O modes in the Y280H mutant at 1966 and 1975 cm(-1). The 1975 cm(-1) mode is assigned to a gamma-form and represents a structure of the active site in which Cu(B) exerts a steric effect on the heme a(3)-bound CO. Therefore, the role of the cross-link is to fix Cu(B) in a certain configuration and distance from heme a(3), and not to allow histidine ligands to coordinate to Cu(B) rather than to heme a(3), rendering the enzyme inactive, as proposed recently (Das, T. K., Pecoraro, C., Tomson, F. L., Gennis, R. B., and Rousseau, D. L. (1998) Biochemistry 37, 14471-14476). The results provide solid evidence that in the Y280H mutant the catalytic site retains its active configuration that allows O(2) binding to heme a(3). Oxygenated intermediates are formed by mixing oxygen with the CO-bound mixed-valence wild type and Y280H enzymes with similar Soret maxima at 438 nm.     

Electron transfer among the CuA-, heme b- and a3-centers of Thermus thermophilus cytochrome ba3

The 1-methyl-nicotinamide radical (MNA(*)), produced by pulse radiolysis has previously been shown to reduce the Cu(A)-site of cytochromes aa(3), a process followed by intramolecular electron transfer (ET) to the heme a but not to the heme a(3) [Farver, O., Grell, E., Ludwig, B., Michel, H. and Pecht, I. (2006) Rates and equilibrium of CuA to heme a electron transfer in Paracoccus denitrificans cytochrome c oxidase. Biophys. J. 90, 2131-2137]. Investigating this process in the cytochrome ba(3) of Thermus thermophilus (Tt), we now show that MNA(*) also reduces Cu(A) with a subsequent ET to the heme b and then to heme a(3), with first-order rate constants 11200 s(-1), and 770 s(-1), respectively. The results provide clear evidence for ET among the three spectroscopically distinguishable centers and indicate that the binuclear a(3)-Cu(B) center can be reduced in molecules containing a single reduction equivalent.     

Stabilization of the peroxy intermediate in the oxygen splitting reaction of cytochrome cbb(3)

The proton-pumping cbb(3)-type cytochrome c oxidases catalyze cell respiration in many pathogenic bacteria. For reasons not yet understood, the apparent dioxygen (O(2)) affinity in these enzymes is very high relative to other members of the heme-copper oxidase (HCO) superfamily. Based on density functional theory (DFT) calculations on intermediates of the oxygen scission reaction in active-site models of cbb(3)- and aa(3)-type oxidases, we find that a transient peroxy intermediate (I(P), Fe[III]-OOH(-)) is ~6kcal/mol more stable in the former case, resulting in more efficient kinetic trapping of dioxygen and hence in a higher apparent oxygen affinity. The major molecular basis for this stabilization is a glutamate residue, polarizing the proximal histidine ligand of heme b(3) in the active site.     

Structural changes that occur upon photolysis of the Fe(II)(a3)-CO complex in the cytochrome ba(3)-oxidase of Thermus thermophilus: a combined X-ray crystallographic and infrared spectral study demonstrates CO binding to Cu(B)

The purpose of the work was to provide a crystallographic demonstration of the venerable idea that CO photolyzed from ferrous heme-a(3) moves to the nearby cuprous ion in the cytochrome c oxidases. Crystal structures of CO-bound cytochrome ba(3)-oxidase from Thermus thermophilus, determined at ~2.8-3.2? resolution, reveal a Fe-C distance of ~2.0?, a Cu-O distance of 2.4? and a Fe-C-O angle of ~126°. Upon photodissociation at 100K, X-ray structures indicate loss of Fe(a3)-CO and appearance of Cu(B)-CO having a Cu-C distance of ~1.9? and an O-Fe distance of ~2.3?. Absolute FTIR spectra recorded from single crystals of reduced ba(3)-CO that had not been exposed to X-ray radiation, showed several peaks around 1975cm(-1); after photolysis at 100K, the absolute FTIR spectra also showed a significant peak at 2050cm(-1). Analysis of the 'light' minus 'dark' difference spectra showed four very sharp CO stretching bands at 1970cm(-1), 1977cm(-1), 1981cm(-1), and 1985cm(-1), previously assigned to the Fe(a3)-CO complex, and a significantly broader CO stretching band centered at ~2050cm(-1), previously assigned to the CO stretching frequency of Cu(B) bound CO. As expected for light propagating along the tetragonal axis of the P4(3)2(1)2 space group, the single crystal spectra exhibit negligible dichroism. Absolute FTIR spectrometry of a CO-laden ba(3) crystal, exposed to an amount of X-ray radiation required to obtain structural data sets before FTIR characterization, showed a significant signal due to photogenerated CO(2) at 2337cm(-1) and one from traces of CO at 2133cm(-1); while bands associated with CO bound to either Fe(a3) or to Cu(B) in "light" minus "dark" FTIR difference spectra shifted and broadened in response to X-ray exposure. In spite of considerable radiation damage to the crystals, both X-ray analysis at 2.8 and 3.2? and FTIR spectra support the long-held position that photolysis of Fe(a3)-CO in cytochrome c oxidases leads to significant trapping of the CO on the Cu(B) atom; Fe(a3) and Cu(B) ligation, at the resolutions reported here, are otherwise unaltered.     

Ligand migration and binding in the dimeric hemoglobin of Scapharca inaequivalvis

Using Fourier transform infrared (FTIR) spectroscopy combined with temperature derivative spectroscopy (TDS) at cryogenic temperatures, we have studied CO binding to the heme and CO migration among cavities in the interior of the dimeric hemoglobin of Scapharca inaequivalvis (HbI) after photodissociation. By combining these studies with X-ray crystallography, three transient ligand docking sites were identified: a primary docking site B in close vicinity to the heme iron, and two secondary docking sites C and D corresponding to the Xe4 and Xe2 cavities of myoglobin. To assess the relevance of these findings for physiological binding, we also performed flash photolysis experiments on HbICO at room temperature and equilibrium binding studies with dioxygen. Our results show that the Xe4 and Xe2 cavities serve as transient docking sites for unbound ligands in the protein, but not as way stations on the entry/exit pathway. For HbI, the so-called histidine gate mechanism proposed for other globins appears as a plausible entry/exit route as well.     

Over-expression of cbaAB genes of Bacillus stearothermophilus produces a two-subunit SoxB-type cytochrome c oxidase with proton pumping activity

We constructed expression plasmids containing cbaAB, the structural genes for the two-subunit cytochrome bo(3)-type cytochrome c oxidase (SoxB type) recently isolated from a Gram-positive thermophile Bacillus stearothermophilus. B. stearothermophilus cells transformed with the plasmids over-expressed an enzymatically active bo(3)-type cytochrome c oxidase protein composed of the two subunits, while the transformed Escherichia coli cells produced an inactive protein composed of subunit I without subunit II. The oxidase over-expressed in B. stearothermophilus was solubilized and purified. The oxidase contained protoheme IX and heme O, as the main low-spin heme and the high-spin heme, respectively. Analysis of the substrate specificity indicated that the high-affinity site is very specific for cytochrome c-551, a cytochrome c that is a membrane-bound lipoprotein of thermophilic Bacillus. The purified enzyme reconstituted into liposomal vesicles with cytochrome c-551 showed H(+) pumping activity, although the efficiency was lower than those of cytochrome aa(3)-type oxidases belonging to the SoxM-type.     

A novel double heme substitution produces a functional bo3 variant of the quinol oxidase aa3 of Bacillus cereus. Purification and paratial characterization

A novel bo3-type quinol oxidase was highly purified from Bacillus cereus PYM1, a spontaneous mutant unable to synthesize heme A and therefore spectroscopically detectable cytochromes aa3 and caa3. The purified enzyme contained 12.4 nmol of heme O and 11.5 nmol of heme B mg-1 protein. The enzyme was composed of two subunits with an Mr of 51,000 and 30,000, respectively. Both subunits were immunoreactive to antibodies raised against the B cereus aa3 oxidase. Moreover, amino-terminal sequence analysis of the 30-kDa subunit revealed that the first 19 residues were identical to those from the 30-kDa subunit of the B. cereus aa3 oxidase. The purified bo3 oxidase failed to oxidize ferrrocytochrome c (neither yeast nor horse) but oxidized tetrachlorohydroquinol with an apparent Km of 498 microM, a Vmax of 21 micromol of O2 min-1mg-1, and a calculated turnover of 55 s-1. The quinol oxidase activity with tetrachlorohydroquinol was inhibited by potassium cyanide and 2-n-heptyl 4-hydroxyquinoline-N-oxide with an I50 of 24 and 300 microM, respectively. Our results demonstrate that the bo3 oxidase of this mutant is not the product of a new operon but instead is a cytochrome aa3 apoprotein encoded by the qox operon of the aa3 oxidase of B. cereus wild type promiscuously assembled with hemes B and O replacing heme A, producing a novel bo3 cytochrome. This is the first reported example of an enzymatically active promiscuous oxidase resulting from the simultaneous substitution of its original hemes in the high and low spin sites.