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.     

Structure of the soluble domain of cytochrome c(552) from Paracoccus denitrificans in the oxidized and reduced states

The crystal structure of the soluble domain of the membrane bound cytochrome c(552) (cytochrome c(552)') from Paracoccus denitrificans was determined using the multiwavelength anomalous diffraction technique and refined at 1.5 A resolution for the oxidized and at 1. 4 A for the reduced state. This is the first high-resolution crystal structure of a cytochrome c at low ionic strength in both redox states. The atomic model allowed for a detailed assessment of the structural properties including the secondary structure, the heme geometry and interactions, and the redox-coupled structural changes. In general, the structure has the same features as that of known eukaryotic cytochromes c. However, the surface properties are very different. Cytochrome c(552)' has a large strongly negatively charged surface part and a smaller positively charged area around the solvent-exposed heme atoms. One of the internal water molecules conserved in all structures of eukaryotic cytochromes c is also present in this bacterial cytochrome c. It contributes to the interactions between the side-chain of Arg36 and the heme propionate connected to pyrrole ring A. Reduction of the oxidized crystals does not influence the conformation of cytochrome c(552)' in contrast to eukaryotic cytochromes c. The oxidized cytochrome c(552)', especially the region of amino acid residues 40 to 56, appears to be more flexible than the reduced one.     

The structure of Paracoccus denitrificans cytochrome c550.

The crystal structure of Paracoccus (formerly Micrococcus) denitrificans cytochrome c550 has been solved by x-ray diffraction to a resolution of 2.45 A. In both amino acid sequence and molecular structure it is evolutionarily homologous with mitochondrial cytochrome c from eukaryotes and photosynthetic cytochrome c2 from purple non-sulfur bacteria. All of these cytochromes c have the same basic folding pattern, with surface insertions of extra amino acids in c550. Various strains of c2 have all, some, or none of the extra insertions observed in c550. The hydrophobic heme environment, position of aromatic rings, and structure and environment of the heme crevice, are virtually identical in cytochromes c55o, c, and c2. Radical changes observed at all regions on the molecular surface except the heme crevice argue for the importance of the crevice and the exposed edge of the heme in the transfer of electrons to and from the cytochrome molecule.     

The cytochrome c peroxidase of Paracoccus denitrificans.

The size, visible absorption spectra, nature of haem and haem content suggest that the cytochrome c peroxidase of Paracoccus denitrificans is related to that of Pseudomonas aeruginosa. However, the Paracoccus enzyme shows a preference for cytochrome c donors with a positively charged 'front surface' and in this respect resembles the cytochrome c peroxidase from Saccharomyces cerevisiae. Paracoccus cytochrome c-550 is the best electron donor tested and, in spite of an acidic isoelectric point, has a markedly asymmetric charge distribution with a strongly positive 'front face'. Mitochondrial cytochromes c have a much less pronounced charge asymmetry but are basic overall. This difference between cytochrome c-550 and mitochondrial cytochrome c may reflect subtle differences in their electron transport roles. A dendrogram of cytochrome c1 sequences shows that Rhodopseudomonas viridis is a closer relative of mitochondria than is Pa. denitrificans. Perhaps a mitochondrial-type cytochrome c peroxidase may be found in such an organism.     

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, c552, acts as the electron mediator between the cytochrome bc1 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 c552, 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 c552 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 c1 gene replacement mutants.

We describe the construction and characterization of gene replacement mutants for the respiratory chain component cytochrome c1 in the bacterium Paracoccus denitrificans. Its structural gene (fbcC) was inactivated by insertion of the kanamycin resistance gene, introduced into a suicide vector, and conjugated into Paracoccus; chromosomal mutants obtained by homologous recombination were selected by antibiotic resistance screening and further characterized biochemically. They showed the complete spectral, enzymatic, and immunological loss of the fbcC gene product together with a serious defect in the assembly of the two other gene products of the fbc operon, cytochrome b and the FeS protein. A possible role of the cytochrome c1 in the assembly process for the enzyme complex is discussed. A functional restoration to wild-type phenotype was achieved by complementing in trans with a newly constructed broad-host-range vector carrying the fbcC gene cassette. When the complete fbc operon was present on this vector, overexpression of complex III subunits was observed. Apart from their physiological significance, such mutants are a prerequisite for probing structure-function relationships by site-directed mutagenesis in order to understand molecular details of electron transport and energy transduction processes of this respiratory enzyme in bacteria and in mitochondria.     

Amino acid sequence of Paracoccus denitrificans cytochrome c550.

The amino acid sequence of Paracoccus (formerly Micrococcus) denitrificans cytochrome c550 has been established by a combination of standard chemical techniques and interpretation of a 2.5 A resolution x-ray electron density map. Peptides derived from a trypsin digest were chemically sequenced, and then ordered by fitting them to the density map. The amino acid compositions of chymotryptic peptides confirmed the x-ray map ordering the tryptic peptides. The amino acid sequence of this respiratory, prokaryotic cytochrome with 134 residues is discussed in relation to those of eukaryotic respiratory cytochrome c (103 to 113 amino acids), and prokaryotic, photosynthetic c2 (103 to 124 amino acids). At the primary structure level, c and c550 differ no more from cytochromes c2 than the various cytochromes c2 do from one another. It is suggested that the respiratory electron transport chain in prokaryotes and eukaryotes is a relatively late evolutionary offshoot of the photosynthetic electron transport chain in purple non-sulfur bacteria.     

Turnover of cytochrome c oxidase from Paracoccus denitrificans

The heme aa3 type cytochrome oxidase from Paracoccus denitrificans incorporated into vesicles with phospholipid reacts during turnover much as the oxidase from mitochondria does. The spectrophotometric changes observed at various wavelengths are closely similar, and the rate is about one-half of that for beef heart oxidase under the same conditions. The rate of appearance of oxidized cytochrome c on initiation of the reaction is also similar and depends on the previous treatment of the oxidase as described by Antonini, E., Brunori, M., Colosimo, A., Greenwood, C. and Wilson, M. T. (1977) Proc. Natl. Acad. Sci. U.S.A. 74, 3128-3132. In terms of their model the resting Paracoccus enzyme is converted to the pulsed form during turnover. The effect is observed with both cytochrome c and hexamine ruthenium as reductants. With the latter a 60-fold increase in rate is observed.     

Reaction of cytochrome c in the electron-transport chain of Paracoccus denitrificans

The nuclear estradiol receptor from chick liver was purified to apparent homogeneity by using a combination of ammonium sulfate precipitation and affinity gel chromatography. The purified receptor migrated as a single band on an SDS-polyacrylamide gel with a molecular weight of 55000 and it exhibited a sedimentation coefficient of 4 S, a dissociation constant of 1.13 nM and a steroid specificity resembling that of the receptor in crude extracts.     

MAD structure of Pseudomonas nautica dimeric cytochrome c552 mimicks the c4 Dihemic cytochrome domain association.

The monohemic cytochrome c552from Pseudomonas nautica (c552-Pn) is thought to be the electron donor to cytochrome cd1, the so-called nitrite reductase (NiR). It shows as high levels of activity and affinity for the P. nautica NiR (NiR-Pn), as the Pseudomonas aeruginosa enzyme (NiR-Pa). Since cytochrome c552is by far the most abundant electron carrier in the periplasm, it is probably involved in numerous other reactions. Its sequence is related to that of the c type cytochromes, but resembles that of the dihemic c4cytochromes even more closely.The three-dimensional structure of P. nautica cytochrome c552has been solved to 2.2 A resolution using the multiple wavelength anomalous dispersion (MAD) technique, taking advantage of the presence of the eight Fe heme ions in the asymmetric unit. Density modification procedures involving 4-fold non-crystallographic averaging yielded a model with an R -factor value of 17.8 % (Rfree=20.8 %). Cytochrome c552forms a tight dimer in the crystal, and the dimer interface area amounts to 19% of the total cytochrome surface area. Four tighly packed dimers form the eight molecules of the asymmetric unit.The c552dimer is superimposable on each domain of the monomeric cytochrome c4from Pseudomomas stutzeri (c4-Ps), a dihemic cytochrome, and on the dihemic c domain of flavocytochrome c of Chromatium vinosum (Fcd-Cv). The interacting residues which form the dimer are both similar in character and position, which is also true for the propionates. The dimer observed in the crystal also exists in solution. It has been hypothesised that the dihemic c4-Ps may have evolved via monohemic cytochrome c gene duplication followed by evolutionary divergence and the adjunction of a connecting linker. In this process, our dimeric c552structure might be said to constitute a "living fossile" occurring in the course of evolution between the formation of the dimer and the gene duplication and fusion. The availability of the structure of the cytochrome c552-Pn and that of NiR from P. aeruginosa made it possible to identify putative surface patches at which the docking of c552to NiR-Pn may occur.     

Proton pump coupled to cytochrome c oxidase in Paracoccus denitrificans

The proton translocating properties of cytochrome c oxidase in whole cells of Paracoccus denitrificans have been studied with the oxidant pulse method. leads to H+/2e- quotients have been measured with endogenous substrates, added methanol and added ascorbate (+TMPD) as reductants, and oxygen and ferricyanide as oxidants. It was found that both the observed leads to H+/O with ascorbate (+TMPD) as reductant, and the differences in proton ejection between oxygen-and ferricyanide pulses, with endogenous substrates or added methanol as a substrate, indicate that the P. denitrificans cytochrome c oxidase translocates protons with a stoichiometry of 2H+/2e-. The results presented in this and previous papers are in good agreement with recent findings concerning the mitochondrial cytochrome c oxidase, and suggest unequal charge separation by different coupling segments of the respiratory chain of P. denitrificans.     

Subunit III of cytochrome c oxidase is expressed in Paracoccus denitrificans

Polyclonal antibodies have been obtained against a synthetic dodecapeptide identical to the aminoacid sequence 120-131 DSPIKDGVWPPE (inferred from its DNA sequence) of Paracoccus denitrificans cytochrome c oxidase subunit III. The antibodies had a titer higher than 1:10000 when tested against the antigen. These antibodies have been used to produce immunological evidence that, despite the fact that subunit III is not isolated with cytochrome c oxidase, it exists in Paracoccus denitrificans lysates. The antibodies did not show reactivity with bovine heart cytochrome c oxidase either by ELISA or immunoblotting. It was also shown that the antibodies react with a single polypeptide present in Paracoccus denitrificans cell lysates, having an apparent molecular weight close to that of subunit III of bovine heart oxidase.     

Reaction of oxygen with cytochrome c oxidase from Paracoccus denitrificans

The reaction of reduced cytochrome c oxidase (EC 1.9.3.1) from Paracoccus denitrificans (American Type Culture Collection 13543) with dioxygen has been followed by laser flash photolysis of the CO derivative. In detergent-stabilized solutions the reaction showed at least two distinct kinetic components, the faster of which was oxygen concentration dependent and had a rate of approximately 60 X 10(6) M-1 s-1. The slower reaction was independent of oxygen concentration and had a rate of 9 X 10(2) s-1. These rates are about 1.5 times greater than comparable rates for ox heart oxidase reported by C. Greenwood and Q. H. Gibson (J. Biol. Chem. (1967) 242, 1782-1787). The kinetic components have markedly different optical spectra which agree precisely in form with those for ox heart enzyme (Greenwood, C., and Gibson, Q. H. (1967) J. Biol. Chem. 242, 1782-1787) but are shifted by 2 nm toward the red. In phospholipid vesicles, the spectral contribution of the faster component was augmented. The dissociation constant for CO at 20 degrees C is 1.6 microM, 6 times greater than for the ox heart enzyme. The bacterial enzyme binds one CO per 2 heme a. The enzyme has an absorption band at 830 nm in the oxidized form similar to that of the ox heart enzyme.     

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

The mechanism of electron coupled proton transfer in cytochrome c oxidase (CcO) 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 H2O2) of the Paraccocus denitrificans CcO 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.     

Solution structure of reduced horse heart cytochrome c

 In the frame of a broad study on the structural differences between the two redox forms of cytochromes to be related to the electron transfer process, the NMR solution structure of horse heart cytochrome c in the reduced form has been determined. The structural data obtained in the present work are compared to those already available in the literature on the same protein and the presence of conformational differences is discussed in the light of the experimental method employed for the structure determination. Redox-state dependent changes are analyzed and in particular they are related to the role of propionate-7 of the heme. Also some hydrogen bonds are changed upon reduction of the heme iron. A substantial similarity is observed for the backbone fold, independently of the oxidation state. At variance, some meaningful differences are observed in the orientation of a few side chains. These changes are related to those found in the case of the highly homologous cytochrome c from Saccharomyces cerevisiae. The exchangeability of the NH protons has been investigated and found to be smaller than in the case of the oxidized protein. We think that this is a characteristic of reduced cytochromes and that mobility is a medium for molecular recognition in vivo. Received: 8 June 1998 / Accepted: 13 October 1998     

Electronic and vibrational spectroscopy of the cytochrome c:cytochrome c oxidase complexes from bovine and Paracoccus denitrificans.

The 1:1 complex between horse heart cytochrome c and bovine cytochrome c oxidase, and between yeast cytochrome c and Paracoccus denitrificans cytochrome c oxidase have been studied by a combination of second derivative absorption, circular dichroism (CD), and resonance Raman spectroscopy. The second derivative absorption and CD spectra reveal changes in the electronic transitions of cytochrome a upon complex formation. These results could reflect changes in ground state heme structure or changes in the protein environment surrounding the chromophore that affect either the ground or excited electronic states. The resonance Raman spectrum, on the other hand, reflects the heme structure in the ground electronic state only and shows no significant difference between cytochrome a vibrations in the complex or free enzyme. The only major difference between the Raman spectra of the free enzyme and complex is a broadening of the cytochrome a3 formyl band of the complex that is relieved upon complex dissociation at high ionic strength. These data suggest that the differences observed in the second derivative and CD spectra are the result of changes in the protein environment around cytochrome a that affect the electronic excited state. By analogy to other protein-chromophore systems, we suggest that the energy of the Soret pi* state of cytochrome a may be affected by (1) changes in the local dielectric, possibly brought about by movement of a charged amino acid side chain in proximity to the heme group, or (2) pi-pi interactions between the heme and aromatic amino acid residues.     

pH-induced conformational transition in the soluble CuA domain of Paracoccus denitrificans cytochrome oxidase

The pH-induced conformational transition in the CuA domain of subunit II of cytochrome oxidase of Paracoccus denitrificans (PdII) has been investigated using various spectroscopic and stopped-flow kinetic methods. UV-visible absorption and circular dichroism studies showed that an increase in pH from 6 to 10 leads to a conformation change with pK(a) = 8.2 associated with the CuA site of the protein. The secondary structure of the protein was, however, shown to remain unchanged in these two conformational states. Thermal and urea-induced unfolding studies showed that the "low-pH" conformation is more stable compared to the "high-pH" conformation of the protein. Moreover, the overall stability of the protein was found to decrease on reduction of the metal centers in the low-pH form, while the oxidation state of the metal centers did not have any significant effect on the overall stability of the protein in the high-pH form. Stopped-flow pH-jump kinetic studies suggested that the conformational transition is associated with a slow deprotonation step followed by fast conformational equilibrium. The results are discussed in the light of understanding the pH-induced conformational change in the beta-barrel structure of the protein and its effect on the coordination geometry of the metal site.     

Mutations in the Ca2+ 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 Ca2+ 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 Ca2+ ligand.