Paracoccus Denitrificans Cytochrome Research Articles

Integral caa3-Cytochrome c Oxidase from Thermus thermophilus: Purification and Crystallization.

Cytochrome c oxidase is a respiratory enzyme catalyzing the energy-conserving reduction of molecular oxygen to water-a fundamental biological process of cell respiration. The first crystal structures of the type A cytochrome c oxidases, bovine heart and Paracoccus denitrificans cytochrome c oxidases, were published in 1995 and contributed immensely to the understanding of the enzyme's mechanism of action. The senior author's research focus was directed toward understanding the structure and function of the type B cytochrome c oxidases, ba3-oxidase and type A2 caa3-oxidase, both from the extreme thermophilic bacterium Thermus thermophilus. While the ba3-oxidase structure was published in 2000 and functional characterization is well-documented in the literature, we recently successfully solved the structure of the caa3-nature made enzyme-substrate complex. This chapter is dedicated to the purification and crystallization process of caa3-cytochrome c oxidase.

The cytochrome ba3 oxidase from Thermus thermophilus does not generate a tryptophan radical during turnover: Implications for the mechanism of proton pumping

Oxygen reduction by cytochrome ba3 oxidase from Thermus thermophilus was studied by stopped-flow and microsecond freeze-hyperquenching analyzed with UV-Vis and EPR spectroscopy. In the initial phase, the low-spin heme b560 is rapidly and almost completely oxidized (kobs>33,000s−1) whereas CuA remains nearly fully reduced. The internal equilibrium between CuA and heme b560 with forward and reverse rate constants of 4621s−1 and 3466s−1, respectively, indicates a ~7.5mV lower midpoint potential for CuA compared to heme b560. The formation of the oxidized enzyme is relatively slow (693s−1). In contrast to the Paracoccus denitrificans cytochrome aa3 oxidase, where in the last phase of the oxidative half cycle a radical from the strictly conserved Trp272 is formed, no radical is formed in the cytochrome ba3 oxidase. Mutation of the Trp229, the cytochrome ba3 oxidase homologue to the Trp272, did not abolish the activity, again in contrast to the Paracoccus cytochrome aa3 oxidase. Differences in the proton pumping mechanisms of Type A and Type B oxidases are discussed in view of the proposed role of the strictly conserved tryptophan residue in the mechanism of redox-linked proton pumping in Type A oxidases. In spite of the differences between the Type A and Type B oxidases, we conclude that protonation of the proton-loading site constitutes the major rate-limiting step in both catalytic cycles.

Electrochemistry suggests proton access from the exit site to the binuclear center in Paracoccus denitrificans cytochrome c oxidase pathway variants

Two different pathways through which protons access cytochrome c oxidase operate during oxygen reduction from the mitochondrial matrix, or the bacterial cytoplasm. Here, we use electrocatalytic current measurements to follow oxygen reduction coupled to proton uptake in cytochrome c oxidase isolated from Paracoccus denitrificans. Wild type enzyme and site-specific variants with defects in both proton uptake pathways (K354M, D124N and K354M/D124N) were immobilized on gold nanoparticles, and oxygen reduction was probed electrochemically in the presence of varying concentrations of Zn2+ ions, which are known to inhibit both the entry and the exit proton pathways in the enzyme. Our data suggest that under these conditions substrate protons gain access to the oxygen reduction site via the exit pathway.

Oxoferryl-Porphyrin Radical Catalytic Intermediate in Cytochrome bd Oxidases Protects Cells from Formation of Reactive Oxygen Species

The quinol-linked cytochrome bd oxidases are terminal oxidases in respiration. These oxidases harbor a low spin heme b(558) that donates electrons to a binuclear heme b(595)/heme d center. The reaction with O(2) and subsequent catalytic steps of the Escherichia coli cytochrome bd-I oxidase were investigated by means of ultra-fast freeze-quench trapping followed by EPR and UV-visible spectroscopy. After the initial binding of O(2), the O-O bond is heterolytically cleaved to yield a kinetically competent heme d oxoferryl porphyrin π-cation radical intermediate (compound I) magnetically interacting with heme b(595). Compound I accumulates to 0.75-0.85 per enzyme in agreement with its much higher rate of formation (~20,000 s(-1)) compared with its rate of decay (~1,900 s(-1)). Compound I is next converted to a short lived heme d oxoferryl intermediate (compound II) in a phase kinetically matched to the oxidation of heme b(558) before completion of the reaction. The results indicate that cytochrome bd oxidases like the heme-copper oxidases break the O-O bond in a single four-electron transfer without a peroxide intermediate. However, in cytochrome bd oxidases, the fourth electron is donated by the porphyrin moiety rather than by a nearby amino acid. The production of reactive oxygen species by the cytochrome bd oxidase was below the detection level of 1 per 1000 turnovers. We propose that the two classes of terminal oxidases have mechanistically converged to enzymes in which the O-O bond is broken in a single four-electron transfer reaction to safeguard the cell from the formation of reactive oxygen species.

Analysis of protein film voltammograms as Michaelis–Menten saturation curves yield the electron cooperativity number for deconvolution

Deconvolution of protein film voltammetric data by fitting multiple components (sigmoids, derivative peaks) often is ambiguous when features are partially overlapping, due to exchangeability between the width and the number of components. Here, a new method is presented to obtain the width of the components. This is based on the equivalence between the sigmoidal catalytic response as function of electrode potential, and the classical saturation curve obtained for the enzyme activity as function of the soluble substrate concentration, which is also sigmoidal when plotted versus log[S]. Thus, analysis of the catalytic voltammogram with Lineweaver–Burk, Eadie–Hofstee, and Hanes–Woolf plots is feasible. This provides a very sensitive measure of the cooperativity number (Hill coefficient), which for electrons equals the apparent (fractional) number of electrons that determine the width, and thereby the number of components (kinetic phases). This analysis is applied to the electrocatalytic oxygen reduction by Paracoccus denitrificans cytochrome aa3 (cytochrome c oxidase). Four partially overlapping kinetic phases are observed that (stepwise) increase the catalytic efficiency with increasingly reductive potential. Translated to cell biology, the activity of the terminal oxidase stepwise adapts to metabolic demand for oxidative phosphorylation.

Cell-free synthesis of cytochrome c oxidase, a multicomponent membrane protein

Cell-free protein synthesis is a useful technique that can site-specifically incorporate isotope-labeled amino acids into proteins. This incorporation is essential for infrared analyses of the electronic state of a specific amino acid residue used to elucidate protein function. Although 17 membrane proteins have been synthesized in their active state by cell-free systems, to date no hetero-subunit protein has been synthesized with this technique, suggesting that there are serious technical limitations. Here we report the cell-free synthesis of Paracoccus denitrificans cytochrome c oxidase, a membrane protein complex composed of three distinct subunits that contain two heme A molecules and two redox-active copper centers. The synthesized protein exhibited normal Soret/vis absorption spectra and ferrocytochrome c oxidation activity.

High resolution crystal structure of Paracoccus denitrificans cytochrome c oxidase: New insights into the active site and the proton transfer pathways

The structure of the two-subunit cytochrome c oxidase from Paracoccus denitrificans has been refined using X-ray cryodata to 2.25 Å resolution in order to gain further insights into its mechanism of action. The refined structural model shows a number of new features including many additional solvent and detergent molecules. The electron density bridging the heme a 3 iron and Cu B of the active site is fitted best by a peroxo-group or a chloride ion. Two waters or OH − groups do not fit, one water (or OH −) does not provide sufficient electron density. The analysis of crystals of cytochrome c oxidase isolated in the presence of bromide instead of chloride appears to exclude chloride as the bridging ligand. In the D-pathway a hydrogen bonded chain of six water molecules connects Asn131 and Glu278, but the access for protons to this water chain is blocked by Asn113, Asn131 and Asn199. The K-pathway contains two firmly bound water molecules, an additional water chain seems to form its entrance. Above the hemes a cluster of 13 water molecules is observed which potentially form multiple exit pathways for pumped protons. The hydrogen bond pattern excludes that the Cu B ligand His326 is present in the imidazolate form.

3P-074 Paracoccus denitrificansチトクロムc酸化酵素の大腸菌由来無細胞系での合成(ヘム蛋白質,第47回日本生物物理学会年会)

3P-074 Paracoccus denitrificansチトクロムc酸化酵素の大腸菌由来無細胞系での合成(ヘム蛋白質,第47回日本生物物理学会年会)

Probing the Paracoccus denitrificans Cytochrome c1−Cytochrome c552 Interaction by Mutagenesis and Fast Kinetics

Electron transfer (ET) between Paracoccus denitrificans cytochrome (cyt) c(1) and cytochrome c(552) was studied using the soluble redox fragments cyt c(1CF) and cyt c(552F). A new ruthenium cyt c(552F) derivative labeled at C23 (Ru(z)-23-c(552F)) was designed to measure rapid electron transfer with cyt c(1CF) in the physiological direction using flash photolysis. The bimolecular rate constant k(12) decreased rapidly with ionic strength above 40 mM, consistent with a diffusional process guided by long-range electrostatic interactions between the two proteins. However, a new kinetic phase was detected at an ionic strength of <35 mM with the ruthenium photoexcitation technique in which k(12) became very rapid (3 x 10(9) M(-1) s(-1)) and nearly independent of ionic strength, suggesting that the reaction became so fast that it was controlled by short-range diffusion along the protein surfaces guided by hydrophobic interactions. These results are consistent with a two-step model for formation of the final encounter complex. No intracomplex electron transfer between Ru(z)-23-c(552F) and c(1CF) was observed even at the lowest ionic strength, indicating that the dissociation constant of the complex was >30 microM. On the other hand, the ruthenium-labeled yeast cytochrome c derivative Ru(z)-39-Cc formed a tight 1:1 complex with cyt c(1CF) at ionic strengths of <60 mM with an intracomplex electron transfer rate constant of 50000 s(-1). A group of cyt c(1CF) variants in the presumed docking site were generated on the basis of information from the yeast cyt bc(1)-cyt c cocrystal structure. Kinetic analysis of cyt c(1CF) mutants located near the heme crevice provided preliminary identification of the interaction site for cyt c(552F) and suggested that formation of the encounter complex is guided primarily by the overall electrostatic surface potential rather than by defined ions.

A D-Pathway Mutation Decouples the Paracoccusdenitrificans Cytochrome c Oxidase by Altering the Side-Chain Orientation of a Distant Conserved Glutamate

Asparagine 131, located near the cytoplasmic entrance of the D-pathway in subunit I of the Paracoccus denitrificans aa(3) cytochrome c oxidase, is a residue crucial for proton pumping. When replaced by an aspartate, the mutant enzyme is completely decoupled: while retaining full cytochrome c oxidation activity, it does not pump protons. The same phenotype is observed for two other substitutions at this position (N131E and N131C), whereas a conservative replacement by glutamine affects both activities of the enzyme. The N131D variant oxidase was crystallized and its structure was solved to 2.32-A resolution, revealing no significant overall change in the protein structure when compared with the wild type (WT), except for an alternative orientation of the E278 side chain in addition to its WT conformation. Moreover, remarkable differences in the crystallographically resolved chain of water molecules in the D-pathway are found for the variant: four water molecules that are observed in the water chain between N131 and E278 in the WT structure are not visible in the variant, indicating a higher mobility of these water molecules. Electrochemically induced Fourier transform infrared difference spectra of decoupled mutants confirm that the protonation state of E278 is unaltered by these mutations but indicate a distinct perturbation in the hydrogen-bonding environment of this residue. Furthermore, they suggest that the carboxylate side chain of the N131D mutant is deprotonated. These findings are discussed in terms of their mechanistic implications for proton routing through the D-pathway of cytochrome c oxidase.

S11/5 The role of the conserved tryptophan272 of the Paracoccus denitrificans cytochrome c oxidase in proton pumping

S11/5 The role of the conserved tryptophan272 of the Paracoccus denitrificans cytochrome c oxidase in proton pumping

S11.23 Synthesis of functional Paracoccusdenitrificanscytochrome c oxidase by Escherichia colicell-free coupled transcription/translation system

S11.23 Synthesis of functional Paracoccusdenitrificanscytochrome c oxidase by Escherichia colicell-free coupled transcription/translation system

The role of the conserved tryptophan272 of the Paracoccus denitrificans cytochrome c oxidase in proton pumping

The catalytic mechanism of heme–copper oxidases – electron transfer coupled to proton pumping – is not yet fully understood. Single turnover experiments in which fully reduced cytochrome aa3 from Paracoccus denitrificans reacts with O2 using the microsecond freeze-hyperquenching sampling technique enabled trapping of transient catalytic intermediates and analysis by low temperature UV–Visible, X-band and Q-band EPR spectroscopy. Our recent findings (Wiertz et al. (2007) J. Biol. Chem. 282, 31580–31591), which show that the strictly conserved W272 is a redox active residue are reviewed here. The W272 forms a tryptophan neutral radical in the transition F→FW⁎→OH in which the novel intermediate FW⁎ harbors the tryptophan radical. The potential role of W272 in proton pumping is highlighted.

Inhibition of bacterial oxidases by formamide and analogs

The enzymatic activity of Paracoccus denitrificans cytochrome c oxidase (COX) and Escherichia coli cytochrome b(o) ubiquinol oxidase (QOX) was determined in the presence of formamide, N,N-dimethyl formamide and N,N-dimethyl acetamide. Formamide was found to inhibit the enzyme activity of the oxidases most significantly, whereas the other two compounds inhibited the activity to a lesser extent. The effects of formamide and analogs on enzyme activity were very similar for COX and QOX, indicating that the mechanism of inhibition might be the same for both of these oxidases. The inhibition kinetics followed a non-competitive mechanism. Studies using proteoliposomes of COX and QOX containing the electron entry site of the enzyme directed towards the outside of the vesicles showed that the effect of inhibition by formamide was higher when the inhibitor was present on the outside of the proteoliposome compared to when it was present only in the aqueous core. This indicates that inhibition of enzyme activity by formamide possibly predominantly involves blocking of the water exit pathway in the oxidases.

Electron transfer kinetics between soluble modules of Paracoccus denitrificans cytochrome c1 and its physiological redox partners

The transient electron transfer (ET) interactions between cytochrome c 1 of the bc 1-complex from Paracoccus denitrificans and its physiological redox partners cytochrome c 552 and cytochrome c 550 have been characterized functionally by stopped-flow spectroscopy. Two different soluble fragments of cytochrome c 1 were generated and used together with a soluble cytochrome c 552 module as a model system for interprotein ET reactions. Both c 1 fragments lack the membrane anchor; the c 1 core fragment ( c 1CF) consists of only the hydrophilic heme-carrying domain, whereas the c 1 acidic fragment ( c 1AF) additionally contains the acidic domain unique to P. denitrificans. In order to determine the ionic strength dependencies of the ET rate constants, an optimized stopped-flow protocol was developed to overcome problems of spectral overlap, heme autoxidation and the prevalent non-pseudo first order conditions. Cytochrome c 1 reveals fast bimolecular rate constants (10 7 to 10 8 M − 1 s − 1 ) for the ET reaction with its physiological substrates c 552 and c 550, thus approaching the limit of a diffusion-controlled process, with 2 to 3 effective charges of opposite sign contributing to these interactions. No direct involvement of the N-terminal acidic c 1-domain in electrostatically attracting its substrates could be detected. However, a slight preference for cytochrome c 550 over c 552 reacting with cyochrome c 1 was found and attributed to the different functions of both cytochromes in the respiratory chain of P. denitrificans.

Kinetic Resolution of a Tryptophan-radical Intermediate in the Reaction Cycle of Paracoccus denitrificans Cytochrome c Oxidase

The catalytic mechanism, electron transfer coupled to proton pumping, of heme-copper oxidases is not yet fully understood. Microsecond freeze-hyperquenching single turnover experiments were carried out with fully reduced cytochrome aa(3) reacting with O(2) between 83 micros and 6 ms. Trapped intermediates were analyzed by low temperature UV-visible, X-band, and Q-band EPR spectroscopy, enabling determination of the oxidation-reduction kinetics of Cu(A), heme a, heme a(3), and of a recently detected tryptophan radical (Wiertz, F. G. M., Richter, O. M. H., Cherepanov, A. V., MacMillan, F., Ludwig, B., and de Vries, S. (2004) FEBS Lett. 575, 127-130). Cu(B) and heme a(3) were EPR silent during all stages of the reaction. Cu(A) and heme a are in electronic equilibrium acting as a redox pair. The reduction potential of Cu(A) is 4.5 mV lower than that of heme a. Both redox groups are oxidized in two phases with apparent half-lives of 57 micros and 1.2 ms together donating a single electron to the binuclear center in each phase. The formation of the heme a(3) oxoferryl species P(R) (maxima at 430 nm and 606 nm) was completed in approximately 130 micros, similar to the first oxidation phase of Cu(A) and heme a. The intermediate F (absorbance maximum at 571 nm) is formed from P(R) and decays to a hitherto undetected intermediate named F(W)(*). F(W)(*) harbors a tryptophan radical, identified by Q-band EPR spectroscopy as the tryptophan neutral radical of the strictly conserved Trp-272 (Trp-272(*)). The Trp-272(*) populates to 4-5% due to its relatively low rate of formation (t((1/2)) = 1.2 ms) and rapid rate of breakdown (t((1/2)) = 60 micros), which represents electron transfer from Cu(A)/heme a to Trp-272(*). The formation of the Trp-272(*) constitutes the major rate-determining step of the catalytic cycle. Our findings show that Trp-272 is a redox-active residue and is in this respect on an equal par to the metallocenters of the cytochrome c oxidase. Trp-272 is the direct reductant either to the heme a(3) oxoferryl species or to Cu (2+)(B). The potential role of Trp-272 in proton pumping is discussed.

Structural and Chemical Changes of the PMIntermediate ofParacoccus denitrificansCytochromecOxidase Revealed by IR Spectroscopy with Labeled Tyrosines and Histidine

Structural and chemical changes in the P(M) intermediate of Paracoccus denitrificans cytochrome c oxidase have been investigated by attenuated total reflection-Fourier transform infrared spectroscopy. Prior studies of P(M) minus oxidized (O) IR difference spectra of unlabeled, universally (15)N-labeled and ring-d(4)-tyrosine-labeled proteins (Iwaki, M., Puustinen, A., Wikström, M., and Rich, P. R. (2004) Biochemistry 43, 14370-14378). provided a basis for band assignments to changes in metal centers and the covalently linked His-Tyr ligand of Cu(B) and highlighted a structural alteration of the protonated Glu278 in the P(M) intermediate. This work has been extended to equivalent measurements on enzymes with (13)C(9)(15)N-labeled and ring-(13)C(6)-labeled tyrosine and with (13)C(6)(15)N(3)-labeled histidine. Histidine labeling allows the assignment of troughs at 1104 and 973 cm(-1) in reduced minus O spectra to histidine changes, whereas tyrosine labeling moves otherwise obscured tyrosine bandshifts to 1454-1437 and 1287-1284 cm(-1). P(M) minus O spectra reveal bands at 1506, 1311, and 1094 cm(-1) in the oxidized state that are replaced by a band at 1519 cm(-1) in P(M). These bands shift with both tyrosine- and histidine-labeling, providing evidence for their assignment to the covalent His-Tyr and for its chemical change in P(M). Comparisons of isotope effects on the amide I regions in P(M) minus O spectra demonstrate that amide carbonyl bonds of tyrosine and histidine are major contributors. This suggests a structural alteration in P(M) that is centered on the His276-Pro277-Glu278-Val279-Tyr280 pentapeptide formed by the His-Tyr covalent linkage. This structural change is proposed to mediate the perturbation of the IR band of the protonated Glu278 headgroup.

Electron transfer among the Cu A-, 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 11 200 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.

Rates and Equilibrium of Cu A to Heme a Electron Transfer in Paracoccus denitrificans Cytochrome c Oxidase

Intramolecular electron transfer between Cu A and heme a in solubilized bacterial (Paracoccus denitrificans) cytochrome c oxidase was investigated by pulse radiolysis. Cu A, the initial electron acceptor, was reduced by 1-methylnicotinamide radicals in a diffusion-controlled reaction, as monitored by absorption changes at 825 nm, followed by partial restoration of the absorption and paralleled by an increase in the heme a absorption at 605 nm. The latter observations indicate partial reoxidation of the Cu A center and the concomitant reduction of heme a. The rate constants for heme a reduction and Cu A reoxidation were identical within experimental error and independent of the enzyme concentration and its degree of reduction, demonstrating that a fast intramolecular electron equilibration is taking place between Cu A and heme a. The rate constants for Cu A → heme a ET and the reverse heme a → Cu A process were found to be 20,400 s −1 and 10,030 s −1, respectively, at 25°C and pH 7.5, which corresponds to an equilibrium constant of 2.0. Thermodynamic and activation parameters of these intramolecular ET reactions were determined. The significance of the results, particularly the low activation barriers, is discussed within the framework of the enzyme’s known three-dimensional structure, potential ET pathways, and the calculated reorganization energies.

The role of tryptophan 272 in the Paracoccus denitrificans cytochrome c oxidase

The mechanism of electron coupled proton transfer in cytochrome c oxidase (C cO) is still poorly understood. The P M-intermediate of the catalytic cycle is an oxoferryl state whose generation requires one additional electron, which cannot be provided by the two metal centres. The missing electron has been suggested to be donated to this binuclear site by a tyrosine residue. A tyrosine radical species has been detected in the P M and F intermediates (formed by addition of H 2O 2) of the Paraccocus denitrificans C cO using electron paramagnetic resonance (EPR) spectroscopy. From the study of conserved variants its origin was determined to be Y167 which is surprising as this residue is not part of the active site. Upon inspection of the active site it becomes evident that W272 could be the actual donor of the missing electron, which can then be replenished from Y167 or from the Y280-H276 cross link in the natural cycle. To address the question, whether such a direct electron transfer pathway to the binuclear centre exists two tryptophan 272 variants in subunit I have been generated. These variants are characterised by their turnover rates as well as using EPR and optical spectroscopy. From these experiments it is concluded, that W272 is an important intermediate in the formation of the radical species appearing in P M and F intermediates produced with hydrogen peroxide. The significance of this finding for the catalytic function of the enzyme is discussed.