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PubMed1997

FnrP and NNR of Paracoccus denitrificans are both members of the FNR family of transcriptional activators but have distinct roles in respiratory adaptation in response to oxygen limitation.

The Paracoccus denitrificans fnrP gene encoding a homologue of the Escherichia coli FNR protein was localized upstream of the gene cluster that encodes the high-affinity cbb3-type oxidase. FnrP harbours the invariant cysteine residues that are supposed to be the ligands of the redox-sensitive [4Fe-4S] cluster in FNR. NNR, another FNR-like transcriptional regulator in P. denitrificans, does not. Analysis of FnrP and NNR single and double mutants revealed that the two regulators each exert exclusive control on the expression of a discrete set of target genes. In FnrP mutants, the expression of cytochrome c peroxidase was blocked, that of membrane-bound nitrate reductase and the cbb3-type oxidase was significantly reduced, whilst the activity of the bb3-type quinol oxidase was increased. The amounts of the nitrite and nitric oxide reductases in these FnrP mutants were the same as in the wild type. NNR mutants, on the other hand, were disturbed exclusively in the concentrations of nitrite reductase and nitric oxide reductase. An FnrP.NNR double mutant combined the phenotypes of the single mutant strains. In all three mutants, the concentrations and/or activities of the aa3-type oxidase, cytochrome C550, cytochrome C552, and nitrous oxide reductase equalled those in the wild type. As the FNR boxes in front of the FnrP- and NNR-regulated genes are highly similar to or even identical to each other, the absence of cross-talk between the regulation by FnrP and NNR implies that as yet unidentified factors are important in the control. It is proposed that the redox state of an intracellular redox couple other than the oxygen/water couple is one of the factors that modulates the activity of FnrP.

Amino Acid Sequence↗

PubMed1997

Paracoccus denitrificans CcmG is a periplasmic protein-disulphide oxidoreductase required for c- and aa3-type cytochrome biogenesis; evidence for a reductase role in vivo.

Cloning and sequencing of the Paracoccus denitrificans ccmG gene indicates that it codes for a periplasmic protein-disulphide oxidoreductase; the presence of the sequence Cys-Pro-Pro-Cys at the CcmG active site suggests that it may act in vivo to reduce disulphide bonds rather than to form them. A CcmG-PhoA fusion confirmed the periplasmic location. Disruption of the ccmG gene resulted in not only the expected phenotype of pleiotropic deficiency in c-type cytochromes, but also loss of spectroscopically detectable cytochrome aa3, cytochrome c oxidase and ascorbate/TMPD oxidase activities; there was also an enhanced sensitivity to growth inhibition by some component of rich media and by oxidized thiol compounds. Dithiothreitol promoted the growth of the ccmG mutant on rich media and substantially restored spectroscopically detectable cytochrome aa3 and cytochrome c oxidase activity, although it did not restore c-type cytochrome biogenesis. Assembly of the disulphide-bridged proteins methanol dehydrogenase and Escherichia coli alkaline phosphatase was unaffected in the ccmG mutant. It is proposed that P. denitrificans CcmG acts in vivo to reduce protein-disulphide bonds in certain protein substrates including c-type cytochrome polypeptides and/or polypeptides involved in c-type cytochrome biogenesis.

Alcohol Oxidoreductases↗

PubMed1999

Transcription regulation of the nir gene cluster encoding nitrite reductase of Paracoccus denitrificans involves NNR and NirI, a novel type of membrane protein.

The nirIX gene cluster of Paracoccus denitrificans is located between the nir and nor gene clusters encoding nitrite and nitric oxide reductases respectively. The NirI sequence corresponds to that of a membrane-bound protein with six transmembrane helices, a large periplasmic domain and cysteine-rich cytoplasmic domains that resemble the binding sites of [4Fe-4S] clusters in many ferredoxin-like proteins. NirX is soluble and apparently located in the periplasm, as judged by the predicted signal sequence. NirI and NirX are homologues of NosR and NosX, proteins involved in regulation of the expression of the nos gene cluster encoding nitrous oxide reductase in Pseudomonas stutzeri and Sinorhizobium meliloti. Analysis of a NirI-deficient mutant strain revealed that NirI is involved in transcription activation of the nir gene cluster in response to oxygen limitation and the presence of N-oxides. The NirX-deficient mutant transiently accumulated nitrite in the growth medium, but it had a final growth yield similar to that of the wild type. Transcription of the nirIX gene cluster itself was controlled by NNR, a member of the family of FNR-like transcriptional activators. An NNR binding sequence is located in the middle of the intergenic region between the nirI and nirS genes with its centre located at position -41.5 relative to the transcription start sites of both genes. Attempts to complement the NirI mutation via cloning of the nirIX gene cluster on a broad-host-range vector were unsuccessful, the ability to express nitrite reductase being restored only when the nirIX gene cluster was reintegrated into the chromosome of the NirI-deficient mutant via homologous recombination in such a way that the wild-type nirI gene was present directly upstream of the nir operon.

Amino Acid Sequence↗

PubMed2002

Two domains of a dual-function NarK protein are required for nitrate uptake, the first step of denitrification in Paracoccus pantotrophus.

Uptake of nitrate into the cytoplasm is the first but least well understood step of denitrification; no gene has previously been identified to be necessary for this process. Upstream from the structural genes of the membrane-bound nitrate reductase (narGHJI) in Paracoccus pantotrophus there is a fusion of two genes, each homologous to members of the narK family. The single open reading frame is predicted to encode 24 transmembrane helices, comprising two domains, NarK1 and NarK2. Analysis of both the accumulation of intracellular nitrite and electron transport through the nitrate reductase enzyme in narK mutants reveals that NarK1 and NarK2 are both involved in nitrate uptake. Maximal rate of nitrate transport via NarK2 was dependent upon nitrite, indicating that NarK2 encodes a nitrate/nitrite antiporter. The uncouplers S13 and dinitrophenol showed that NarK2 was not dependent on the proton motive force for maximal nitrate transport activity. Nitrate transport via NarK1 was dependent on proton motive force, indicating that it is likely to be a nitrate/proton symporter. Low expression of membrane-bound nitrate reductase in narK mutants was counteracted by azide, which induced nitrate reductase expression only if the transcriptional activator NarR was present.

Amino Acid Sequence↗

PubMed2001

New pathway of amine oxidation respiratory chain of Paracoccus denitrificans IFO 12442.

The physiological electron acceptor of quinohemoprotein amine dehydrogenase (QH-AmDH) from Paracoccus denitrificans IFO 12442 was identified by biochemical and electrochemical methods. Of three types of heme c-containing proteins purified together with QH-AmDH from the periplasm of n-butylamine-grown cells, only constitutive cytochrome c-550 was reduced by the addition of QH-AmDH and n-butylamine. Reconstitution of the respiratory chain revealed that cytochrome c-550 mediates the electron transfer from QH-AmDH to the terminal oxidase. This is a new pathway of the amine oxidation respiratory chain of P. denitrificans.

Amines↗

PubMed2002

Mutations in the docking site for cytochrome c on the Paracoccus heme aa3 oxidase. Electron entry and kinetic phases of the reaction.

Introducing site-directed mutations in surface-exposed residues of subunit II of the heme aa3 cytochrome c oxidase of Paracoccus denitrificans, we analyze the kinetic parameters of electron transfer from reduced horse heart cytochrome c. Specifically we address the following issues: (a) which residues on oxidase contribute to the docking site for cytochrome c, (b) is an aromatic side chain required for electron entry from cytochrome c, and (c) what is the molecular basis for the previously observed biphasic reaction kinetics. From our data we conclude that tryptophan 121 on subunit II is the sole entry point for electrons on their way to the CuA center and that its precise spatial arrangement, but not its aromatic nature, is a prerequisite for efficient electron transfer. With different reaction partners and experimental conditions, biphasicity can always be induced and is critically dependent on the ionic strength during the reaction. For an alternative explanation to account for this phenomenon, we find no evidence for a second cytochrome c binding site on oxidase.

Binding Sites↗

PubMed2004

Isolation and biochemical characterization of two soluble iron(III) reductases from Paracoccus denitrificans.

Two soluble enzymes (FerA and FerB) catalyzing the reduction of a number of iron(III) complexes by NADH, were purified to near homogeneity from the aerobically grown iron-limited culture of Paracoccus denitrificans using a combination of anion-exchange chromatography (Sepharose Q), chromatofocusing (Mono P), and gel permeation chromatography (Superose 12). FerA is a monomer with a molecular mass of 19 kDa, whereas FerB exhibited a molecular mass of about 55 kDa and consists of probably two identical subunits. FerA can be classified as an NADH:flavin oxidoreductase with a sequential reaction mechanism. It requires the addition of FMN or riboflavin for activity on Fe(III) substrates. In these reactions, the apparent substrate specificity of FerA seems to stem exclusively from different chemical reactivities of Fe(III) compounds with the free reduced flavin produced by the enzyme. Observations on reducibility of Fe(III) chelated by vicinal dihydroxy ligands support the view that FerA takes part in releasing iron from the catechol type siderophores synthesized by P. denitrificans. Contrary to FerA, the purified FerB contains a noncovalently bound redox-active FAD coenzyme, can utilize NADPH in place of NADH, does not reduce free FMN at an appreciable rate, and gives a ping-pong type kinetic pattern with NADH and Fe(III)-nitrilotriacetate as substrates. FerB is able to reduce chromate, in agreement with the fact that its N-terminus bears a homology to the previously described chromate reductase from Pseudomonas putida. Besides this, it also readily reduces quinones like ubiquinone-0 (Q0) or unsubstituted p-benzoquinone.

Amino Acid Sequence↗

PubMed1998

Cytochrome-c-binding site on cytochrome oxidase in Paracoccus denitrificans.

To monitor the docking site for cytochrome c on cytochrome oxidase from Paracoccus denitrificans, a series of site-directed mutants in acidic residues exposed on the three largest subunits was constructed, and the purified enzymes were assayed for their steady-state kinetic parameters, their ionic strength dependence, and their fast electron entry kinetics by stopped-flow measurements. Increasing the ionic strength, the maximum of the bell-shaped dependence of the steady-state rate observed for wild type shifts the maximum to lower ionic strength in most of the mutants. The Km determined in steady-state experiments under different conditions is largely increased for most of the subunit II and one of the subunit I mutants, giving evidence that binding is impaired, whereas subunit III residues do not seem to contribute significantly. In addition, the bimolecular rate constant for cytochrome c oxidation under pre-steady state conditions was measured using stopped flow spectroscopy. Taken together, the results demonstrate that the initial interaction of cytochrome c and oxidase is mediated through glutamates and aspartates mainly located in subunit II. The crystal structure of oxidase reveals that the participating residues are clustered, creating an extended, negatively charged patch. We propose this clustering to be a decisive factor in the recognition of positively charged patches on the surface of cytochrome c.

Binding Sites↗

PubMed1998

Mutational analysis of residues forming hydrogen bonds in the Rieske [2Fe-2S] cluster of the cytochrome bc1 complex in Paracoccus denitrificans.

Two mutations (S157A and Y159F) in the Rieske iron-sulfur subunit of the ubihydroquinone-cytochrome c oxidoreductase from Paracoccus denitrificans have been characterized with respect to the protein and [2Fe-2S] cluster stability, the enzyme activity and the redox potential of the [2Fe-2S] cluster. In the structure of the water-soluble fragment of the Rieske iron-sulfur protein of the bovine heart mitochondrial bc1 complex, both residues (S163 and Y165 in the bovine sequence) form the following hydrogen bonds to sulfur atoms in the [2Fe-2S] cluster: Ser163 O gamma-S-1 and Tyr165 O eta-Cys139 S gamma [Iwata, S., Saynovits, M., Link, T. A. & Michel, H. (1996) Structure 4, 567-579]. They are conserved in all known Rieske iron-sulfur proteins from bc1 complexes including that from P. denitrificans. Both amino acid exchanges, introduced as separate point mutations on plasmids containing the fbc operon, lead to a fully assembled three-subunit complex in P. denitrificans, with a metal and heme content as well as subunit composition indistinguishable from the parental strain. The purified complexes show decreased turnover numbers and redox potentials of the Rieske cluster. Whereas the turnover number of the bc1 complex isolated from the parental strain was 145 s(-1), the turnover numbers for the mutants S157A and Y159F were 10 s(-1) and 52 s(-1), respectively, corresponding to 7% and 36% activity, respectively. The midpoint potential Em of the Rieske cluster at pH 6 and 5 degrees C was +360 mV for the bc1 complex from the parental strain; the values in the mutant complexes were +316 mV (Y159F) and +265 mV (S157A). Shifts of the g values in the electron paramagnetic resonance spectra indicate an altered electron distribution in the mutants compared to in the wild-type protein.

Amino Acid Sequence↗

PubMed1998

31P-NMR spectroscopy of human and Paracoccus denitrificans electron transfer flavoproteins, and 13C- and 15N-NMR spectroscopy of human electron transfer flavoprotein in the oxidised and reduced states.

Human and Paracoccus denitrificans wild-type electron transfer flavoproteins have been investigated by 31P-NMR in the oxidised and reduced states. The 31P chemical shifts of the diphosphate moiety of the protein-bound FAD were similar in the proteins and were independent of the redox state. The chemical shifts were remarkably similar to those of ferredoxin-NADP+ reductase and, to a lesser degree, with those of NADPH-cytochrome P-450 reductase. The wild-type human electron transfer apoprotein was reconstituted with [2,4a-13C2]FAD, [4,10a-13C2]FAD, or [U-15N4]FAD. The reconstituted proteins were studied by 13C- and 15N-NMR techniques in the oxidised and reduced states. The chemical shifts were compared with those of free flavin in aqueous solution or in chloroform, and those of flavoproteins published in the literature. In the oxidised state, strong hydrogen bonds exist between residues of the apoprotein and C(2)O and N(5) of FAD. The N(1) atom is also hydrogen bonded and, as shown by X-ray data, involves the C'(4)-OH group of FAD. The sp2 hybridisation of N(10) is small compared to other flavoproteins. In the reduced state, there are strong hydrogen bonds involving C(2)O and N(5) of FAD. The N(1) atom is ionised as observed also in other flavoproteins when investigated by NMR. The intramolecular hydrogen bond between the C'(4)-OH group and the N(1) atom of FAD is maintained in the reduced state, suggesting an involvement in the stabilisation of a certain configuration of the diphosphate group of protein-bound FAD in both redox states. The N(10) atom in the reduced protein is highly sp3 hybridised in comparison to those of other flavoproteins.

Apoproteins↗

PubMed1999

The reduction state of the Q-pool regulates the electron flux through the branched respiratory network of Paracoccus denitrificans.

In this work we demonstrate how the reduction state of the Q-pool determines the distribution of electron flow over the two quinol-oxidising branches in Paracoccus denitrificans: one to quinol oxidase, the other via the cytochrome bc1 complex to the cytochrome c oxidases. The dependence of the electron-flow rate to oxygen on the fraction of quinol in the Q-pool was determined in membrane fractions and in intact cells of the wild-type strain, a bc1-negative mutant and a quinol oxidase-negative mutant. Membrane fractions of the bc1-negative mutant consumed oxygen at significant rates only at much higher extents of Q reduction than did the wild-type strain or the quinol oxidase-negative mutant. In the membrane fractions, dependence on the Q redox state was exceptionally strong corresponding to elasticity coefficients close to 2 or higher. In intact cells, the dependence was weaker. In uncoupled cells the dependence of the oxygen-consumption rates on the fractions of quinol in the Q-pool in the wild-type strain and in the two mutants came closer to that found for the membrane fractions. We also determined the dependence for membrane fractions of the wild-type in the absence and presence of antimycin A, an inhibitor of the bc1 complex. The dependence in the presence of antimycin A resembled that of the bc1-negative mutant. These results indicate that electron-flow distribution between the two quinol-oxidising branches in P. denitrificans is not only determined by regulated gene expression but also, and to a larger extent, by the reduction state of the Q-pool.

Electron Transport↗

PubMed2000

Sulfite and membrane energization induce two different active states of the Paracoccus denitrificans F0F1-ATPase.

Activation of the latent ATPase activity of inside-out vesicles from plasma membranes of Paracoccus denitrificans was studied. Several factors were found to induce activation: heat, membrane energization by succinate oxidation, methanol, oxyanions (sulfite, phosphate, arsenate, bicarbonate) and limited proteolysis with trypsin. Among the oxyanions, sulfite induced the higher increase in ATPase activity. Sulfite functioned as a nonessential activator that slightly modified the affinity for ATP and increased notoriously the Vmax. There was a competitive effect between sulfite, bicarbonate and phosphate for ATPase activation; their similar chemical geometry suggests that these oxyanions have a common binding site on the enzyme. Dithiothreitol did not affect the ATPase activity. ATPase activation by sulfite was decreased by uncoupler, enhanced by trypsin and inhibited by ADP, oligomycin and venturicidin. In contrast, activation induced by succinate was less sensitive to ADP, oligomycin, venturicidin and trypsin. It is proposed that the active states induced by sulfite and succinate reflect two conformations of the enzyme, in which the inhibitory subunit epsilon is differently exposed to trypsin.

Adenosine Diphosphate↗

PubMed2000

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.

Amino Acid Sequence↗

PubMed2001

Regulation of expression of terminal oxidases in Paracoccus denitrificans.

In order to study the induction of terminal oxidases in Paracoccus denitrificans, their promoters were fused to the lacZ reporter gene and analysed in the wild-type strain, in an FnrP-negative mutant, in a cytochrome bc1-negative mutant, and in six single or double oxidase-negative mutant strains. The strains were grown under aerobic, semi-aerobic, and denitrifying conditions. The oxygen-sensing transcriptional-regulatory protein FnrP negatively regulated the activity of the qox promoter, which controls expression of the ba3-type quinol oxidase, while it positively regulated the activity of the cco promoter, which controls expression of the cbb3-type cytochrome c oxidase. The ctaDII and ctaC promoters, which control the expression of the aa3-type cytochrome c oxidase subunits I and II, respectively, were not regulated by FnrP. The activities of the latter two promoters, however, did decrease with decreasing oxygen concentrations in the growth medium, suggesting that an additional oxygen-sensing mechanism exists that regulates transcription of ctaDII and ctaC. Apparently, the intracellular oxygen concentration (as sensed by FnrP) was not the only signal to which the oxidase promoters responded. At given extracellular oxygen status, both the qox and the cco promoters responded to mutations in terminal oxidase genes, whereas the ctaDII and ctaC promoters did not. The change of electron distribution through the respiratory network, resulting from elimination of one or more oxidase genes, may have changed intracellular signals that affect the activities of the qox and cco promoters. On the other hand, the re-routing of electron distribution in the respiratory mutants hardly affected the oxygen consumption rate as compared to that of the wild-type. This suggests that the mutants adapted their respiratory network in such a way that they were able to consume oxygen at a rate similar to that of the wild-type strain.

Base Sequence↗

PubMed2001

The peroxidase activity of cytochrome c-550 from Paracoccus versutus.

Next to their natural electron transport capacities, c-type cytochromes possess low peroxidase and cytochrome P-450 activities in the presence of hydrogen peroxide. These catalytic properties, in combination with their structural robustness and covalently bound cofactor make cytochromes c potentially useful peroxidase mimics. This study reports on the peroxidase activity of cytochrome c-550 from Paracoccus versutus and the loss of this activity in presence of H2O2. The rate-determining step in the peroxidase reaction of cytochrome c-550 is the formation of a reactive intermediate, following binding of peroxide to the haem iron. The reaction rate is very low compared to horseradish peroxidase (approximately one millionth), because of the poor accessibility of the haem iron for H2O2, and the lack of a base catalyst such as the distal His of the peroxidases. This is corroborated by the linear dependence of the reaction rate on the peroxide concentration up to at least 1 M H2O2. Steady-state conversion of a reducing substrate, guaiacol, is preceded by an activation phase, which is ascribed to the build-up of amino-acid radicals on the protein. The inactivation kinetics in the absence of reducing substrate are mono-exponential and shown to be concurrent with haem degradation up to 25 mM H2O2 (pH 8.0). At still higher peroxide concentrations, inactivation kinetics are biphasic, as a result of a remarkable protective effect of H2O2, involving the formation of superoxide and ferrocytochrome c-550.

Amino Acids↗

PubMed2000

Single-electron reduction of the oxidized state is coupled to proton uptake via the K pathway in Paracoccus denitrificans cytochrome c oxidase.

The reductive part of the catalytic cycle of cytochrome c oxidase from Paracoccus denitrificans was examined by using time-resolved potential measurements on black lipid membranes. Proteoliposomes were adsorbed to the black lipid membranes and Ru(II)(2, 2'-bipyridyl)(3)(2+) was used as photoreductant to measure flash-induced membrane potential generation. Single-electron reduction of the oxidized wild-type cytochrome c oxidase reveals two phases of membrane potential generation (tau(1) approximately 20 micros and tau(2) approximately 175 micros) at pH 7.4. The fast phase is not sensitive to cyanide and is assigned to electron transfer from Cu(A) to heme a. The slower phase is inhibited completely by cyanide and shows a kinetic deuterium isotope effect by a factor of 2-3. Although two enzyme variants mutated in the so-called D pathway of proton transfer (D124N and E278Q) show the same time constants and relative amplitudes as the wild-type enzyme, in the K pathway variant K354M, tau(2) is increased to 900 micros. This result suggests uptake of a proton through the K pathway during the transition from the oxidized to the one-electron reduced state. After the second laser flash under anaerobic conditions, a third electrogenic phase with a time constant of approximately 1 ms appears. The amplitude of this phase grows with increasing flash number. We explain this growth by injection of a second electron into the single-electron reduced enzyme. On multiple flashes, both D pathway mutants behave differently compared with the wild type and two additional slow phases of tau(3) approximately 2 ms and tau(4) approximately 15 ms are observed. These results suggest that the D pathway is involved in proton transfer coupled to the uptake of the second electron.

Amino Acid Substitution↗

PubMed1988

Cytochrome c oxidase from Paracoccus denitrificans: both hemes are located in subunit I.

The two-subunit cytochrome c oxidase from Paracoccus denitrificans has been sequentially digested with chymotrypsin and Staphylococcus aureus V8 protease. The smaller subunit of the enzyme (apparent Mr 32,000) was split into numerous peptides that were removed by anion-exchange HPLC. The larger subunit was only digested to a limited extent (from an apparent Mr 45,000 to Mr 43,000), and the spectral properties were preserved relative to the native enzyme (a reduced minus oxidized difference spectrum with maxima at 447 and 607 nm in the Soret and alpha region, respectively). As judged from CO-reduced spectra this proteolytically digested, one-fragment oxidase was found to contain an equal amount of cytochromes a and a3. The enzymatic activity with reduced cytochrome c as substrate in the presence of Triton X-100 proceeded with equal affinity (apparent Km = 0.5-1.0 microM) and with a Vmax of approximately 20% (40 s-1) of that found with the native enzyme (200 s-1). When the assay system was supplemented with soybean phospholipids, the Km became 2 microM for both enzymes and the Vmax became 730 and 170 s-1 for the native and the digested enzyme, respectively. Thus subunit I of P. denitrificans oxidase, and most probably of the other cytochrome c oxidases as well, contains both hemes and at least one Cu atom and has significant enzymatic activity.

Chymotrypsin↗

PubMed1997

Structure at 2.7 A resolution of the Paracoccus denitrificans two-subunit cytochrome c oxidase complexed with an antibody FV fragment.

The aa3 type cytochrome c oxidase consisting of the core subunits I and II only was isolated from the soil bacterium Paracoccus denitrificans and crystallized as complex with a monoclonal antibody Fv fragment. Crystals could be grown in the presence of a number of different nonionic detergents. However, only undecyl-beta-D-maltoside and cyclohexyl-hexyl-beta-D-maltoside yielded well-ordered crystals suitable for high resolution x-ray crystallographic studies. The crystals belong to space group P212121 and diffract x-rays to at least 2.5 A (1 A = 0.1 nm) resolution using synchrotron radiation. The structure was determined to a resolution of 2.7 A using molecular replacement and refined to a crystallographic R-factor of 20.5% (Rfree = 25.9%). The refined model includes subunits I and II and the 2 chains of the Fv fragment, 2 heme A molecules, 3 copper atoms, and 1 Mg/Mn atom, a new metal (Ca) binding site, 52 tentatively identified water molecules, and 9 detergent molecules. Only four of the water molecules are located in the cytoplasmic half of cytochrome c oxidase. Most of them are near the interface of subunits I and II. Several waters form a hydrogen-bonded cluster, including the heme propionates and the Mg/Mn binding site. The Fv fragment binds to the periplasmic polar domain of subunit II and is critically involved in the formation of the crystal lattice. The crystallization procedure is well reproducible and will allow for the analysis of the structures of mechanistically interesting mutant cytochrome c oxidases.

Binding Sites↗