Paracoccus Denitrificans Cytochrome Research Articles

S1f1-4 Cell-free synthesis of functional Paracoccus denitrificans cytochrome c oxidase(S1-f1: "Structural chemical studies on physiological functions of proteins",Symposia,Abstract,Meeting Program of EABS & BSJ 2006)

S1f1-4 Cell-free synthesis of functional Paracoccus denitrificans cytochrome c oxidase(S1-f1: "Structural chemical studies on physiological functions of proteins",Symposia,Abstract,Meeting Program of EABS & BSJ 2006)

Is a third proton-conducting pathway operative in bacterial cytochrome c oxidase?

Despite the existence of several three-dimensional structures of cytochrome c oxidases, a detailed understanding of pathways involved in proton movements through the complex remains largely elusive. Next to the two well-established pathways (termed D and K), an additional proton-conducting network ('H-channel') has been proposed for the beef heart enzyme. Yet, our recent mutational studies on corresponding residues of the Paracoccus denitrificans cytochrome c oxidase provide no clues that such a pathway operates in the prokaryotic enzyme.

Probing the access of protons to the K pathway in the Paracoccus denitrificans cytochrome c oxidase

In recent studies on heme-copper oxidases a particular glutamate residue in subunit II has been suggested to constitute the entry point of the so-called K pathway. In contrast, mutations of this residue (E78(II)) in the Paracoccus denitrificans cytochrome c oxidase do not affect its catalytic activity at all (E78(II)Q) or reduce it to about 50% (E78(II)A); in the latter case, the mutation causes no drastic decrease in heme a(3) reduction kinetics under anaerobic conditions, when compared to typical K pathway mutants. Moreover, both mutant enzymes retain full proton-pumping competence. While oxidized-minus-reduced Fourier-transform infrared difference spectroscopy demonstrates that E78(II) is indeed addressed by the redox state of the enzyme, absence of variations in the spectral range characteristic for protonated aspartic and glutamic acids at approximately 1760 to 1710 cm(-1) excludes the protonation of E78(II) in the course of the redox reaction in the studied pH range, although shifts of vibrational modes at 1570 and 1400 cm(-1) reflect the reorganization of its deprotonated side chain at pH values greater than 4.8. We therefore conclude that protons do not enter the K channel via E78(II) in the Paracoccus enzyme.

ATR-FTIR Spectroscopy and Isotope Labeling of the PM Intermediate of Paracoccus denitrificans Cytochrome c Oxidase

The structure of the P(M) intermediate of Paracoccus denitrificans cytochrome c oxidase was investigated by perfusion-induced attenuated total reflection-Fourier transform infrared (ATR-FTIR) spectroscopy. Transitions from the oxidized to P(M) state were initiated by perfusion with CO/oxygen buffer, and the extent of conversion was quantitated by simultaneously monitoring visible absorption changes. In prior work, tentative assignments of bands were proposed for heme a(3), a change in the environment of the protonated state of a carboxylic acid, and a covalently linked histidine-tyrosine ligand to Cu(B) that has been found in the catalytic site. In this work, reduced minus oxidized difference spectra at pH 6.5 and 9.0 and P(M) minus oxidized difference spectra at pH 9.0 were compared in unlabeled, universally (15)N-labeled, and tyrosine-ring-d(4)-labeled proteins to improve these assignments. In the reduced minus oxidized difference spectrum, (15)N labeling resulted in large changes in the amide II region and a 9 cm(-1) downshift in a 1105 cm(-1) trough that is attributed to histidine. In contrast, changes induced by tyrosine-ring-d(4) labeling were barely detectable where the isotope-sensitive bands are expected. Both isotope substitutions had large effects on P(M) minus oxidized difference spectra. A prominent trough at 1542 cm(-1) was shifted to 1527 cm(-1) with (15)N labeling, and its magnitude was diminished with the appearance of a 1438 cm(-1) trough with tyrosine-ring-d(4) labeling. Both isotope substitutions also had large effects on a 1314 cm(-1) trough in the same spectra. These shifts indicate that the bands are linked to both a nitrogenous compound and a tyrosine, the most obvious candidate being the covalent histidine-tyrosine ligand of Cu(B). Comparison with model material data suggests that the tyrosine hydroxyl group is protonated when the binuclear center is oxidized but deprotonated in the P(M) intermediate. Positive bands at 1519 and 1570 cm(-1) were replaced with bands at 1504 and 1556 cm(-1), respectively, with tyrosine-ring-d(4) labeling, are characteristic of upsilon(7a)(C-O) and upsilon(C-C) bands of neutral phenolic radicals, and most likely reflect the formation of the neutral radical state of the histidine-tyrosine ligand in P(M).

ATR-FTIR spectroscopy of the P(M) and F intermediates of bovine and Paracoccus denitrificans cytochrome c oxidase.

The structures of P(M) and F intermediates of bovine and Paracoccus denitrificans cytochrome c oxidase were investigated by perfusion-induced attenuated total reflection-Fourier transform infrared (ATR-FTIR) spectroscopy. Transitions from the "fast" oxidized state to the P(M) or F states were initiated by perfusion with buffer containing either CO/oxygen or H(2)O(2). Intermediates were quantitated by simultaneous monitoring of visible absorption changes in the protein film. For both bovine and P. denitrificans oxidase, the major features of the IR difference spectrum of P(M) were similar when produced by CO/oxygen or by H(2)O(2) treatments. These IR difference spectra were distinctly different from the IR difference spectrum of F that formed with extended treatment with H(2)O(2). Some IR bands could be assigned tentatively to perturbations of heme a(3) ring modes and substituents, and these perturbations were greater in P(M) than in F. Other bands could be assigned to surrounding protein changes. Strong perturbation of the environment of a carboxylic acid, most likely E-242 (bovine numbering), occurred in P(M) and relaxed back in F. A second redox-sensitive carboxylic acid was also perturbed in the bovine P(M) intermediate. Further consistent signatures of P(M) in both oxidases that were absent in F were strong negative bands at 1547 and 1313 cm(-1) in bovine oxidase (1542 and 1314 cm(-1) in P. denitrificans) and a positive band at approximately 1519 cm(-1). From comparison with available IR data on model compounds, it is suggested that these reflect changes in the covalent tyrosine-histidine ligand to Cu(B). These findings are discussed in relation to the oxidase catalytic cycle.

Nitric Oxide Reacts with the Single-electron Reduced Active Site of Cytochrome c Oxidase

The reduction kinetics of the mutants K354M and D124N of the Paracoccus denitrificans cytochrome oxidase (heme aa(3)) by ruthenium hexamine was investigated by stopped-flow spectrophotometry in the absence/presence of NO. Quick heme a reduction precedes the biphasic heme a(3) reduction, which is extremely slow in the K354M mutant (k(1) = 0.09 +/- 0.01 s(-1); k(2) = 0.005 +/- 0.001 s(-1)) but much faster in the D124N aa(3) (k(1) = 21 +/- 6 s(-1); k(2) = 2.2 +/- 0.5 s(-1)). NO causes a very large increase (>100-fold) in the rate constant of heme a(3) reduction in the K354M mutant but only a approximately 5-fold increase in the D124N mutant. The K354M enzyme reacts rapidly with O(2) when fully reduced but is essentially inactive in turnover; thus, it was proposed that impaired reduction of the active site is the cause of activity loss. Since at saturating [NO], heme a(3) reduction is approximately 100-fold faster than the extremely low turnover rate, we conclude that, contrary to O(2), NO can react not only with the two-electron but also with the single-electron reduced active site. This mechanism would account for the efficient inhibition of cytochrome oxidase activity by NO in the wild-type enzyme, both from P. denitrificans and from beef heart. Results also suggest that the H(+)-conducting K pathway, but not the D pathway, controls the kinetics of the single-electron reduction of the active site.

Backbone dynamics of the functional domain of Paracoccus denitrificans Cytochrome c552 in the Oxidized state

Backbone dynamics of the functional domain of Paracoccus denitrificans Cytochrome c552 in the Oxidized state

Solution structure and dynamics of the functional domain of Paracoccus denitrificans cytochrome c(552) in both redox states.

A soluble and fully functional 10.5 kDa fragment of the 18.2 kDa membrane-bound cytochrome c(552) from Paracoccus denitrificans has been heterologously expressed and (13)C/(15)N-labeled to study the structural features of this protein in both redox states. Well-resolved solution structures of both the reduced and oxidized states have been determined using high-resolution heteronuclear NMR. The overall protein topology consists of two long terminal helices and three shorter helices surrounding the heme moiety. No significant redox-induced structural differences have been observed. (15)N relaxation rates and heteronuclear NOE values were determined at 500 and 600 MHz. Several residues located around the heme moiety display increased backbone mobility in both oxidation states, while helices I, III, and V as well as the two concatenated beta-turns between Leu30 and Arg36 apparently form a less flexible domain within the protein structure. Major redox-state-dependent differences of the internal backbone mobility on the picosecond-nanosecond time scale were not evident. Hydrogen exchange experiments demonstrated that the slow-exchanging amide proton resonances mainly belong to the helices and beta-turns, corresponding to the regions with high order parameters in the dynamics data. Despite this correlation, the backbone amide protons of the oxidized cytochrome c(552) exchange considerably faster with the solvent compared to the reduced protein. Using both differential scanning calorimetry as well as temperature-dependent NMR spectroscopy, a significant difference in the thermostabilities of the two redox states has been observed, with transition temperatures of 349.9 K (76.8 degrees C) for reduced and 307.5 K (34.4 degrees C) for oxidized cytochrome c(552). These results suggest a clearly distinct backbone stability between the two oxidation states.

Zn 2+ binding to the cytoplasmic side of Paracoccus denitrificans cytochrome c oxidase selectively uncouples electron transfer and proton translocation

Using a combination of stopped-flow spectrophotometric proton pumping measurements and time-resolved potential measurements on black lipid membranes, we have investigated the effect of Zn 2+ ions on the proton transfer properties of Paracoccus denitrificans cytochrome c oxidase. When zinc was enclosed in the interior of cytochrome c oxidase containing liposomes, the H/ e stoichiometry was found to gradually decrease with increasing Zn 2+ concentration. Half-inhibition of proton pumping was observed at [Zn 2+] i =75 μM corresponding to about 5–6 Zn 2+ ions per oxidase molecule. In addition, there was a significant increase in the respiratory control ratio of the proteoliposomes upon incorporation of Zn 2+. Time-resolved potential measurements on a black lipid membrane showed that the electrogenic phases slowed down in the presence of Zn 2+ correspond to phases that have been attributed to proton uptake from the cytoplasmic side and to proton pumping. We conclude that Zn 2+ ions bind close to or within the two proton transfer pathways of the bacterial cytochrome c oxidase.

Heme-copper oxidases with modified D- and K-pathways are yet efficient proton pumps

The cytochrome aa 3-type quinol oxidase from the archaeon Acidianus ambivalens and the ba 3 -type cytochrome c oxidase from Thermus thermophilus are divergent members of the heme-copper oxidase superfamily of enzymes. In particular they lack most of the key residues involved in the proposed proton transfer pathways. The pumping capability of the A. ambivalens enzyme was investigated and found to occur with the same efficiency as the canonical enzymes. This is the first demonstration of pumping of 1 H +/electron in a heme-copper oxidase that lacks most residues of the K- and D-channels. Also, the structure of the ba 3 oxidase from T. thermophilus was simulated by mutating Phe274 to threonine and Glu278 to isoleucine in the D-pathway of the Paracoccus denitrificans cytochrome c oxidase. This modification resulted in full efficiency of proton translocation albeit with a substantially lowered turnover. Together, these findings show that multiple structural solutions for efficient proton conduction arose during evolution of the respiratory oxidases, and that very few residues remain invariant among these enzymes to function in a common proton-pumping mechanism.

Solution structure of the functional domain of Paracoccus denitrificans cytochrome c552 in the reduced state.

In order to determine the solution structure of Paracoccus denitrificans cytochrome c552 by NMR, we cloned and isotopically labeled a 10.5-kDa soluble fragment (100 residues) containing the functional domain of the 18.2-kDa membrane-bound protein. Using uniformly 15N-enriched samples of cytochrome c552 in the reduced state, a variety of two-dimensional and three-dimensional heteronuclear double-resonance NMR experiments was employed to achieve complete 1H and 15N assignments. A total of 1893 distance restraints was derived from homonuclear 2D-NOESY and heteronuclear 3D-NOESY spectra; 1486 meaningful restraints were used in the structure calculations. After restrained energy minimization a family of 20 structures was obtained with rmsd values of 0.56 +/- 0. 10 A and 1.09 +/- 0.09 A for the backbone and heavy atoms, respectively. The overall topology is similar to that seen in previously reported models of this class of proteins. The global fold consists of two long helices at the N-terminus and C-terminus and three shorter helices surrounding the heme moiety; the helices are connected by well-defined loops. Comparison with the X-ray structure shows some minor differences in the positions of the Trp57 and Phe65 side-chain rings as well as the heme propionate groups.

Mutation of Arg-54 Strongly Influences Heme Composition and Rate and Directionality of Electron Transfer in Paracoccus denitrificans Cytochrome c Oxidase

The effect of a single site mutation of Arg-54 to methionine in Paracoccus denitrificans cytochrome c oxidase was studied using a combination of optical spectroscopy, electrochemical and rapid kinetics techniques, and time-resolved measurements of electrical membrane potential. The mutation resulted in a blue-shift of the heme a alpha-band by 15 nm and partial occupation of the low-spin heme site by heme O. Additionally, there was a marked decrease in the midpoint potential of the low-spin heme, resulting in slow reduction of this heme species. A stopped-flow investigation of the reaction with ferrocytochrome c yielded a kinetic difference spectrum resembling that of heme a(3). This observation, and the absence of transient absorbance changes at the corresponding wavelength of the low-spin heme, suggests that, in the mutant enzyme, electron transfer from Cu(A) to the binuclear center may not occur via heme a but that instead direct electron transfer to the high-spin heme is the dominating process. This was supported by charge translocation measurements where Deltapsi generation was completely inhibited in the presence of KCN. Our results thus provide an example for how the interplay between protein and cofactors can modulate the functional properties of the enzyme complex.

Mutations in the Ca 2+ binding site of the Paracoccus denitrificans cytochrome c oxidase

Recent structure determinations suggested a new binding site for a non-redox active metal ion in subunit I of cytochrome c oxidase both of mitochondrial and of bacterial origin. We analyzed the relevant metal composition of the bovine and the Paracoccus denitrificans enzyme and of bacterial site-directed mutants in several residues presumably liganding this ion. Unlike the mitochondrial enzyme where a low, substoichiometric content of Ca 2+ was found, the bacterial wild-type (WT) oxidase showed a stoichiometry of one Ca per enzyme monomer. Mutants in Asp-477 (in immediate vicinity of this site) were clearly diminished in their Ca content and the isolated mutant enzyme revealed a spectral shift in the heme a visible absorption upon Ca addition, which was reversed by Na ions. This spectral behavior, largely comparable to that of the mitochondrial enzyme, was not observed for the bacterial WT oxidase. Further structure refinement revealed a tightly bound water molecule as an additional Ca 2+ ligand.

Heterologous expression of soluble fragments of cytochrome c552 acting as electron donor to the Paracoccus denitrificans cytochrome c oxidase

A membrane-bound c-type cytochrome, c 552, acts as the electron mediator between the cytochrome bc 1 complex and cytochrome c oxidase in the branched respiratory chain of the bacterium Paracoccus denitrificans. Unlike in mitochondria where a soluble cytochrome c interacts with both complexes, the bacterial c 552, the product of the cycM gene, shows a tripartite structure, with an N-terminal membrane anchor separated from a typical class I cytochrome domain by a highly charged region. Two derivative fragments, lacking either only the membrane spanning region or both N-terminal domains, were constructed on the genetic level, and expressed in Escherichia coli cotransformed with the ccm gene cluster encoding host-specific cytochrome c maturation factors. High levels of cytochromes c were expressed and located in the periplasm as holo-proteins; both these purified c 552 fragments are functional in electron transport to oxidase, as ascertained by kinetic measurements, and will prove useful for future structural studies of complex formation by NMR and X-ray diffraction.

Paracoccus denitrificans cytochrome c oxidase: a kinetic study on the two- and four-subunit complexes

Cytochrome c oxidase from Paracoccus denitrificans has been purified in two different forms differing in polypeptide composition. An enzyme containing polypeptides I–IV is obtained when the purification procedure is performed in β- d-dodecylmaltoside. If, however, Triton X-100 is used to purify the enzyme under otherwise identical conditions, an enzyme is obtained containing only subunits I–II. The two enzymes are undistinguishable by optical spectroscopy but show significant differences in the transient and steady-state time regimes, as studied by stopped-flow spectroscopy. The observed differences, however, are not due to removal of subunits III and IV, but rather to a specific effect of Triton X-100 which appears to affect cytochrome c binding. From these results it is not expected that subunits III and IV play any significant role in cytochrome c binding and, possibly, in the subsequent electron transfer processes. The results also suggest that both electrostatic and hydrophobic interactions may be important in the initial electron transfer process from cytochrome c.

The Coupling of Electron Transfer and Proton Translocation: Electrostatic Calculations on Paracoccus denitrificans Cytochrome c Oxidase

We have calculated the electrostatic potential and interaction energies of ionizable groups and analyzed the response of the protein environment to redox changes in Paracoccus denitrificans cytochrome c oxidase by using a continuum dielectric model and finite difference technique. Subsequent Monte Carlo sampling of protonation states enabled us to calculate the titration curves of all protonatable groups in the enzyme complex. Inclusion of a model membrane allowed us to restrict the calculations to the functionally essential subunits I and II. Some residues were calculated to have complex titration curves, as a result of strong electrostatic coupling, desolvation, and dipolar interactions. Around the heme a 3-Cu B binuclear center, we have identified a cluster of 18 strongly interacting residues that account for most of the proton uptake linked to electron transfer. This was calculated to be between 0.7 and 1.1 H + per electron, depending on the redox transition considered. A hydroxide ion bound to Cu B was determined to become protonated to form water upon transfer of the first electron to the binuclear site. The bulk of the protonation changes linked to further reduction of the heme a 3-Cu B center was calculated to be due to proton uptake by the interacting cluster and Glu II-78. Upon formation of the three-electron reduced state (P1), His 325, modeled in an alternative orientation away from Cu B, was determined to become protonated. The agreement of these results with experiment and their relevance in the light of possible mechanisms of redox-coupled proton transfer are discussed.

Probing the ground state of the purple mixed valence CuA center in nitrous oxide reductase: a CW ENDOR (X-band) study of the 65Cu, 15N-histidine labeled enzyme and interpretation of hyperfine couplings by molecular orbital calculations

CW ENDOR (X-band) spectra for the purple mixed-valence [Cu(1.5+)...Cu(1.5+)], S = 1/2, CuA site in nitrous oxide reductase were obtained after insertion of 65Cu or both 65Cu and 15N-histidine. The 14N/15N isotopic substitution allowed for an unambiguous deconvolution of proton and nitrogen hyperfine couplings in the spectra. A single nitrogen coupling with a value of 12.9 ± 0.4 MHz for 14N was detected. Its anisotropy was characteristic for imidazole bound to copper. A spin density of 3–5% was estimated for the nitrogen donors to CuA, indicating that the ground state is 2B3u. Proton hyperfine structure was detected from four Cβ protons of coordinating cysteine residues. Their isotropic and anisotropic parts were deconvoluted by spectral simulation. From the anisotropic couplings a spin density of 16–24% was estimated for each of the cysteine thiolate donors of CuA. The [NHisCu(RS)2CuNHis]+ core structure of CuA in nitrous oxide reductase from Pseudomonas stutzeri is predicted to be similar to the crystallographically determined CuA* structure (Wilmanns M, Lappalainen P, Kelly M, Sauer-Eriksson E, Saraste M (1995) Proc Natl Acad Sci USA 92 : 11955–11959), but distinct from the CuA structure of Paracoccus denitrificans cytochrome c oxidase (Iwata S, Ostermeier C, Ludwig B, Michel H (1995) Nature 376 : 660–669). The angular dependence of the isotropic couplings as a function of the electronic ground state was calculated by the INDO/S method. The Mulliken atomic-spin populations calculated by a gradient-corrected density functional method and the semiempirical INDO/S method were compared with experimentally derived spin populations, and good agreement between theory and experiment was found for both calculations. The ground state of CuA is best represented by the resonance structures of the form [CuIS–S–CuII↔ CuIS•S–CuI↔ CuIS–S•CuI↔ CuIIS–S–CuI]. It is proposed that the Cu 4s,p as well as sulfur 3d orbitals play a role in the stabilization of this novel type of cluster.

The role of electrostatic interactions for cytochrome c oxidase function.

In recent years, the enormous increase in high-resolution three-dimensional structures of proteins together with the development of powerful theoretical techniques have provided the basis for a more detailed examination of the role of electrostatics in determining the midpoint potentials of redox-active metal centers and in influencing the protonation behavior of titratable groups in proteins. Based on the coordinates of the Paracoccus denitrificans cytochrome c oxidase, we have determined the electrostatic potential in and around the protein, calculated the titration curves for all ionizable residues in the protein, and analyzed the response of the protein environment to redox changes at the metal centers. The results of this study provide insight into how charged groups can be stabilized within a low-dielectric environment and how the range of their electrostatic effects can be modulated by the protein. A cluster of 18 titratable groups around the heme a3-CuB binuclear center, including a hydroxide ion bound to the copper, was identified that accounts for most of the proton uptake associated with redox changes at the binuclear site. Predicted changes in net protonation were in reasonable agreement with experimentally determined values. The relevance of these findings in the light of possible mechanisms of redox-coupled proton movement is discussed.

Asp-193 and Glu-218 of subunit II are involved in the Mn 2+-binding of Paracoccus denitrificans cytochrome c oxidase

Cytochrome c oxidase contains a binding site for a non-redox-active metal at the interface of subunits I and II, usually a magnesium ion. In Paracoccus denitrificans oxidase, typically 20% may be replaced by manganese, using standard growth media. Site-directed mutants were constructed in subunit II (D193N and E218Q), and the isolated enzymes analyzed by total-reflection X-ray fluorescence spectrometry and EPR. Both mutants show a strong reduction of the manganese stoichiometry and a diminished electron transfer activity, demonstrating that D193 and E218 are involved in the binding of a manganese/magnesium ion in this site.

Carboxyl group protonation upon reduction of the Paracoccus denitrificans cytochrome c oxidase: direct evidence by FTIR spectroscopy

The redox reactions of the cytochrome c oxidase from Paracoccus denitrificans were investigated in a thin-layer cell designed for the combination of electrochemistry under anaerobic conditions with UVIVIS and IR spectroscopy. Quantitative and reversible electrochemical reactions were obtained at a surface-modified electrode for all cofactors as indicated by the optical signals in the 400–700 nm range. Fourier transform infrared (FTIR) difference spectra of reduction and oxidation (reduced-minus-oxidized and oxidized-minus-reduced, respectively) obtained in the 1800-1000 cm −1 range reveal highly structured band features with major contributions in the amide I (1620–1680 cm −1) and amide II (1580-1520 cm −1) range which indicate structural rearrangements in the cofactor vicinity. However, the small amplitude of the IR difference signals indicates that these conformational changes are small and affect only individual peptide groups. In the spectral region above 1700 cm −1, a positive peak in the reduced state (1733 cm −1) and negative peak in the oxidized state (1745 cm −1) are characteristic for the formation and decay of a COOH mode upon reduction. The most obvious interpretation of this difference signal is proton uptake by one Asp or Gin side chain carboxyl group in the reduced state and deprotonation of another Asp or Glu residue. Moreover, both residues could well be coupled as a donor-acceptor pair in the proton transfer chain. An alternative interpretation is in terms of a protonated carboxyl group which shifts to a different environment in the reduced state. The relevance of this first direct observation of protein protonation changes in the cytochrome c oxidase for vectorial proton transfer and the catalytic reaction is discussed.