The role of blue copper proteins in the oxidation of methylamine by an obligate methylotroph.

Organism 4025, an obligate methylotroph, when grown on methylamine in the presence of a high concentration of copper, contained high concentrations of methylamine dehydrogenase and two blue copper proteins, amicyanin and an azurin-type protein; these were purified to homogeneity and characterized. The methylamine dehydrogenase is a basic protein (pI 8.8) and consists of light and heavy subunits (Mr 14100 and 43000; total Mr 112000). This dehydrogenase differed slightly from other methylamine dehydrogenases in its absorption spectrum and in its lack of thermal stability. Amicyanin, the more abundant blue copper protein, had an Mr of 11500, a midpoint redox potential of 294mV at pH 7.0, and a much lower isoelectric point (pI5.3) than other amicyanins. Its absorption maximum was 620 nm (7-24 nm higher than those of other amicyanins); its absorption coefficient (at 620 nm) was 3.8 mM-1 X cm-1. The 'azurin' (6% of the blue copper protein) had an Mr of 12500, a midpoint redox potential of 323 mV and a high isoelectric point (pI 9.4). Its absorption maximum was 620 nm, the absorption coefficient (16 mM-1 X cm-1) at this wavelength being considerably greater than that of any blue copper protein described previously. The partially-purified soluble cytochromes cH and cL were similar to those of other methylotrophs. The interactions of the purified redox proteins were investigated in order to elucidate their role in methylamine oxidation. Methylamine dehydrogenase was able to donate electrons only to amicyanin, the rate of reaction being 2.04 mmol/min per mumol of methylamine dehydrogenase; this is sufficient to account for the rate of respiration in whole bacteria. The blue copper proteins were able to react rapidly with each other and with both the soluble cytochromes c.     

Characterization of a novel soluble c-type cytochrome in a moxD mutant of Methylobacterium extorquens AM1

Methylobacterium extorquens AM1 contains a novel c-type cytochrome, called cytochrome c-553, previously thought to be a precursor of the electron acceptor (cytochrome cL) for methanol dehydrogenase. Its amino acid composition and serological characteristics show that it has no structural relationship to cytochrome cL. It usually comprises less than 5% of the total c-type cytochromes. In a moxD mutant, which contains neither methanol dehydrogenase nor cytochrome cL, it comprises 30% of the soluble cytochrome and it has been purified and characterized from that mutant. Cytochrome c-553 is large (Mr 23,000), acidic and monohaem, with a redox potential of 194 mV. It reacts rapidly and completely with CO but is not autoxidizable. It is not autoreducible, and it is not an electron acceptor from methanol dehydrogenase or methylamine dehydrogenase, nor an important electron donor to the oxidase. It is able to accept electrons from cytochrome cL and to donate electrons to cytochrome cH. It is present in the soluble fraction (presumably periplasmic) and membrane fraction of wild-type bacteria during growth on a wide range of growth substrates, but its function in these bacteria or in the moxD mutant has not been determined.     

The methylamine oxidizing system of Pseudomonas AM1 reconstituted with purified components

The electron transport system coupled to the oxidation of methylamine in Pseudomonas AM1 was investigated by reconstituting it from the highly purified components. A mixture of methylamine dehydrogenase, cytochrome cH and cytochrome c oxidase (= cytochrome aa3) actively oxidized methylamine (161 mol of O2 consumed/mol of heme a of cytochrome c oxidase X min). In this system, addition of amicyanin did not affect the oxygen consumption rate. The oxygen consumption rate of the cell-free extract prepared from the cells cultivated in a copper-deficient medium was directly proportional to the amount of amicyanin added, and extrapolation to zero copper concentration gave a value of 28 mol of O2 consumed/mol of heme a of cytochrome c oxidase X min. These results suggest that methylamine oxidation in the bacterium can occur at least to some extent without participation of amicyanin.     

Cytochromes c550, c552, and c1 in the Electron Transport Network of Paracoccus denitrificans: Redundant or Subtly Different in Function?

Paracoccus denitrificans strains with mutations in the genes encoding the cytochrome c(550), c(552), or c(1) and in combinations of these genes were constructed, and their growth characteristics were determined. Each mutant was able to grow heterotrophically with succinate as the carbon and free-energy source, although their specific growth rates and maximum cell numbers fell variably behind those of the wild type. Maximum cell numbers and rates of growth were also reduced when these strains were grown with methylamine as the sole free-energy source, with the triple cytochrome c mutant failing to grow on this substrate. Under anaerobic conditions in the presence of nitrate, none of the mutant strains lacking the cytochrome bc(1) complex reduced nitrite, which is cytotoxic and accumulated in the medium. The cytochrome c(550)-deficient mutant did denitrify provided copper was present. The cytochrome c(552) mutation had no apparent effect on the denitrifying potential of the mutant cells. The studies show that the cytochromes c have multiple tasks in electron transfer. The cytochrome bc(1) complex is the electron acceptor of the Q-pool and of amicyanin. It is also the electron donor to cytochromes c(550) and c(552) and to the cbb(3)-type oxidase. Cytochrome c(552) is an electron acceptor both of the cytochrome bc(1) complex and of amicyanin, as well as a dedicated electron donor to the aa(3)-type oxidase. Cytochrome c(550) can accept electrons from the cytochrome bc(1) complex and from amicyanin, whereas it is also the electron donor to both cytochrome c oxidases and to at least the nitrite reductase during denitrification. Deletion of the c-type cytochromes also affected the concentrations of remaining cytochromes c, suggesting that the organism is plastic in that it adjusts its infrastructure in response to signals derived from changed electron transfer routes.     

The methanol-oxidizing system of Methylobacterium extorquens AM1 reconstituted with purified constituents

The electron transport system (with cytochrome aa3) coupled to the oxidation of methanol in Methylobacterium extorquens AM1 (former Pseudomonas AM1) was reconstituted with highly purified constituents of the system. A mixture of 2.7 microM methanol dehydrogenase, 3.2 microM cytochrome cH, and 71 nM cytochrome c oxidase (= cytochrome aa3) consumed oxygen at a lower rate in the presence of methanol, while its activity was enhanced 3-fold by the addition of 1.4 microM cytochrome cL (74 mol of O2 consumed/mol of heme a of cytochrome c oxidase per min). Further addition of amicyanin to the above mixture did not affect the activity. Although ammonium ion greatly activated the activity of methanol dehydrogenase, the ion had little effect on the oxygen consumption activity of the above mixture. On the basis of the results obtained in the present study, an electron transport system is proposed for the oxidation of methanol in M. extorquens AM1.     

Amicyanin: an electron acceptor of methylamine dehydrogenase

A type I blue copper protein, “amicyanin”, was purified from a cell-free extract of methylamine-grown Pseudomonas AM1. It was found that amicyanin is able to serve as a primary electron acceptor of methylamine dehydrogenase. Amicyanin was reduced by the addition of both methylamine dehydrogenase and methylamine. Cytochromes c could not be directly reduced but could be reduced with the addition of amicyanin. The results strongly suggest that amicyanin participates as an electron carrier between methylamine dehydrogenase and cytochrome c in the electron transport chain of the methylamine-grown cell.     

The autoreducible cytochromes c of the methylotrophs Methylophilus methylotrophus and Pseudomonas AM1.

The two types of soluble cytochrome c (cytochrome cH and cytochrome cL) found in methylotrophs are completely distinct proteins; one type is not a dimer or degradation product of the other. Free thiol groups are probably not involved in the unusually rapid autoreduction of the cytochromes at high pH. The axial ligands to the haem iron, histidine and methionine, are the same as in other low-spin cytochromes c. The methionine ligand is displaced at high pH by an alternative strong-field ligand. This displacement does not occur on reduction of cytochrome cL by methanol dehydrogenase, but this does not rule out the possibility that the autoreduction mechanism is involved in the interaction of the dehydrogenase and cytochrome c.     

The interaction between methanol dehydrogenase and the autoreducible cytochromes c of the facultative methylotroph Pseudomonas AM1.

Cytochromes cH and cL were autoreduced at high pH (pK greater than 10) and the autoreduced cytochromes reacted with CO. The autoreduction was first-order with respect to oxidized cytochrome c and was reversible by lowering the pH. Pure methanol dehydrogenase reduced cytochrome c (in the absence of methanol) by lowering the pK for autoreduction to less than 8.5. A mechanism is proposed for the autoreduction of cytochrome c and its involvement in the reaction with methanol dehydrogenase.     

The significance of the flexible loop in the azurin (Az-iso2) from the obligate methylotroph Methylomonas sp. strain J

The obligate methylotroph Methylomonas sp. strain J produces two azurins (Az-iso1 and Az-iso2) as candidates for electron acceptor from methylamine dehydrogenase (MADH) in the electron-transfer process involving the oxidation of methylamine to formaldehyde and ammonia. The X-ray crystallographic study indicated that Az-iso2 gives two types of crystals (form I and form II) with polyethylene glycol (PEG4000) and ammonium sulfate as the precipitants, respectively. Comparison between the two Az-iso2 structures in forms I and II reveals the remarkable structural changes at the top surface of the molecule around the copper atom. Az-iso2 possesses Gly43 instead of Val43 or Ala43, which is unique among all other azurins around the copper ligand His46, inducing the remarkable structural change in the loop region from Gly37 to Gly43. When the structure of Az-iso2 is superimposed on that of amicyanin in the ternary complex composed of MADH, amicyanin, and cytochrome c(551), the loop of Az-iso2 deeply overlaps with the light subunit of MADH. However, the Az-iso2 molecule is probably able to avoid any steric hindrance with the cognate MADH to form the complex for intermolecular electron-transfer reaction, since the loop containing Gly43 is flexible. We discuss why the electron-transfer activity of Az-iso2 is fivefold higher than that of Az-iso1.     

Characterisation of the electron transport chain of an obligate methylotroph,strain 4025

• 1.1. The obligate methanol-utilising bacterium strain 4025 contains cytochromes b and c. Cytochrome a is never present.
• 2.2. The soluble cytochrome c is similar to that from other methylotrophs in reacting (slowly) with carbon monoxide and it can be separated into two types, differing markedly in their isoelectric points.
• 3.3. Some of the cytochrome b reacts rapidly with carbon monoxide and is thus the likely cytochrome oxidase (cytochrome o).
• 4.4. The partially purified, NAD⁺-independent methanol dehydrogenase is similar to such enzymes from the other methanol-utilising bacteria in respect of its prosthetic group, dependence on ammonia or methylamine for activity and its wide substrate specificity.
• 5.5. The fluorescence seen in colonies of this organism is probably due to a flavin derivative.
• 6.6. This study of electron transport components does not shed any light on the unusually high copper requirement shown by this methylotroph.

     

An engineered CuA Amicyanin capable of intermolecular electron transfer reactions

The type I copper center of amicyanin was replaced with a binuclear CuA center. To create this model CuA protein, a portion of the amino acid sequence that contains three of the ligands to the native type I copper center of Paracoccus denitrificans amicyanin was replaced with the corresponding portion of sequence that provides five ligands for the CuA center of cytochrome c oxidase from P. denitrificans. UV-visible and electron paramagnetic resonance spectroscopy confirm that the engineered protein as isolated possesses the mixed-valence Cu1.5Cu1.5 (purple) CuA center. Comparison of the spectroscopic properties of this CuA amicyanin with those of the CuA centers of other natural and engineered CuA proteins suggests that the spectroscopic features may be dictated more by the protein host than the sequence of the CuA loop. Novel reactions for a simple CuA model protein are also described. In contrast to other natural and engineered CuA proteins, the fully reduced CuA amicyanin may be reoxidized by molecular oxygen to the mixed-valence state. It is also shown that CuA amicyanin can serve as an electron donor and an electron acceptor for other redox proteins. The mixed-valence form accepts electrons from cytochromes c-551i and c-550 from P. denitrificans. The fully reduced form donates electrons to native and P94F amicyanin. The function as either an electron donor or acceptor is consistent with the measured redox potential of CuA amicyanin of +273 mV. These data indicate that this CuA amicyanin will be a particularly useful model protein for structure-function studies of reactivity and the electron transfer properties of the CuA redox center.     

Cloning, sequencing and expression studies of the genes encoding amicyanin and the beta-subunit of methylamine dehydrogenase from Thiobacillus versutus.

The genes encoding amicyanin and the beta-subunit of methylamine dehydrogenase (MADH) from Thiobacillus versutus have been cloned and sequenced. The organization of these genes makes it likely that they are coordinately expressed and it supports earlier findings that the blue copper protein amicyanin is involved in electron transport from methylamine to oxygen. The amino acid sequence deduced from the nucleotide sequence of the amicyanin-encoding gene is in agreement with the published protein sequence. The gene codes for a pre-protein with a 25-amino-acid-long signal peptide. The amicyanin gene could be expressed efficiently in Escherichia coli. The protein was extracted with the periplasmic fraction, indicating that pre-amicyanin is translocated across the inner membrane of E. coli. Sequence studies on the purified beta-subunit of MADH confirm the amino acid sequence deduced from the nucleotide sequence of the corresponding gene. The latter codes for a pre-protein with an unusually long (56 amino acids) leader peptide. The sequencing results strongly suggest that pyrroloquinoline quinone (PQQ) or pro-PQQ is not the co-factor of MADH.     

The molecular structure of an unusual cytochrome c2 determined at 2.0 A; the cytochrome cH from Methylobacterium extorquens.

Cytochrome cH is the electron donor to the oxidase in methylotrophic bacteria. Its amino acid sequence suggests that it is a typical Class 1 cytochrome c, but some features of the sequence indicated that its structure might be of special interest. The structure of oxidized cytochrome cH has been solved to 2.0 A resolution by X-ray diffraction. It has the classical tertiary structure of the Class 1 cytochromes c but bears a closer gross resemblance to mitochondrial cytochrome c than to the bacterial cytochrome c2. The left-hand side of the haem cleft is unique; in particular, it is highly hydrophobic, the usual water is absent, and the "conserved" Tyr67 is replaced by tryptophan. A number of features of the structure demonstrate that the usual hydrogen bonding network involving water in the haem channel is not essential and that other mechanisms may exist for modulation of redox potentials in this cytochrome.     

Site-directed mutagenesis of proline 94 to alanine in amicyanin converts a true electron transfer reaction into one that is kinetically coupled

Amicyanin is a type I copper protein that mediates electron transfer (ET) from methylamine dehydrogenase (MADH) to cytochrome c-551i. Pro(94) resides in the "ligand loop" of amicyanin, a sequence of amino acids that contains three of the four copper ligands. ET from the reduced O-quinol tryptophan tryptophylquinone of MADH to oxidized P94A amicyanin is a true ET reaction that exhibits values of electronic coupling (H(AB)) and reorganization energy (lambda) that are the same as for the reaction of native amicyanin. In contrast, the parameters for the ET reaction from reduced P94A amicyanin to oxidized cytochrome c-551i have been significantly altered as a consequence of the mutation. These values of H(AB) and lambda are 8.3 cm(-)(1) and 2.3 eV, respectively, compared to values of 0.3 cm(-)(1) and 1.2 eV for the reaction of native reduced amicyanin. The crystal structure of reduced P94A amicyanin exhibits two alternate conformations with the positions of the copper 1.4 A apart [Carrell, C. J., Sun, D., Jiang, S., Davidson, V. L., and Mathews, F. S. (2004) Biochemistry 43, 9372-9380]. In one of these, conformation B, a water molecule has replaced Met(98) as a copper ligand, and the ET distance to the heme of the cytochrome is increased by 1.4 A. Analysis of these structures suggests that the true k(ET) for ET from the copper in conformation B to heme would be much less than for ET from conformation A. A novel kinetic mechanism is proposed to explain these data in which the reduction of Cu(2+) by methylamine dehydrogenase is a true ET reaction while the oxidation of Cu(1+) by cytochrome c-551i is kinetically coupled ET. By comparison of the temperature dependence of the observed rate of the coupled ET reaction from reduced P94A amicyanin to cytochrome c-551i with the predicted rates and temperature dependence for the true ET reaction from conformation A, it was possible to determine the K(eq) and values of DeltaH degrees and DeltaS degrees that are associated with the non-ET reaction that modulates the observed ET rate.     

The interaction of methanol dehydrogenase and its electron acceptor, cytochrome cL in methylotrophic bacteria.

The interactions of methanol dehydrogenase (MDH, EC1.1.99.8) with its specific electron acceptor cytochrome cL has been investigated in Methylobacterium extorquens and Methylophilus methylotrophus. The MDHs of these two very different methylotrophs have the same alpha 2 beta 2 structure; the interaction of these MDHs with their specific electron acceptor, cytochrome cL, has been studied using a novel assay system. Electrostatic reactions are involved in 'docking' of the two proteins. EDTA inhibits the reaction by a process involving neither metal chelation nor the 'docking' process. Chemical modification studies showed that the two proteins interact by a 'docking' process involving interactions of lysyl residues on MDH and carboxyl residues on cytochrome cL. When 'zero length', two stage cross-linking was done (with proteins from both bacteria), the alpha-subunits of MDH cross-linked with cytochrome cL by way of lysyl groups on MDH and carboxyl groups on the cytochrome. Tuna mitochondrial cytochrome c provided a model for cytochrome cH which is the electron acceptor for cytochrome cL in the 'methanol oxidase' electron transport chain. Tuna cytochrome c was shown to form crosslinked products with carboxyl-modified cytochrome cL. MDH and tuna cytochrome c competed for the same domain on cytochrome cL. It was concluded that MDH reacts with cytochrome cL by an electrostatic reaction which involves carboxyl groups on cytochrome cL and amino groups on the alpha-subunit of MDH. The same domain on cytochrome cL is involved in subsequent 'docking' with its electron acceptor.     

Methylobacillus flagellatus KT contains a novel cbo-type cytochrome oxidase

The o-type oxidase from the methanol-grown obligate methylotroph Methylobacillus flagellatus KT has been purified to homogeneity. The complex is composed of four subunits (57, 40, 35 and 30 kDa). It contains six haems (4C:1B:1O) and one copper atom per molecule. It is proposed that the haem O-Cu(B) binuclear centre and a low-spin haem B are located in subunit I (57 kDa), two haems C reside in the cytochrome c homodimer (35 kDa), two haems C belong to the dihaem cytochrome c (30 kDa). The presented data provide evidence that cytochrome cbo is a novel representative of the haem-copper oxidase superfamily.     

The soluble cytochromes c of methanol-grown Hyphomicrobium X. Evidence against the involvement of autoreduction in electron-acceptor functioning of cytochrome cL.

Hyphomicrobium X, grown on methanol with O2 or nitrate as electron acceptor, contains two major soluble cytochromes c. These were isolated in electrophoretically homogeneous form. They are related to cytochromes c already described for other methylotrophic bacteria and designated cytochromes cH and cL (properties indicated in that order) in view of the following characteristics: absorption maxima of the reduced forms (414, 520 and 551 nm and 414, 520 and 550 nm); molar absorption coefficients of the alpha-bands (23,700 M-1.cm-1 and 21,600 M-1.cm-1); maxima of the alpha-bands (no splitting) at 77 K (547.6 nm and 548.5 nm); Mr values of the native proteins (15,000 and 19,500); pI values (7.4 and 7.5, and 4.3); midpoint potentials at pH 7.0 (+292 mV and +270 mV). Both were monomers containing 1 haem c group per protein molecule, the oxidized forms binding cyanide at high pH. Autoreduction also occurred at high pH but at a rate significantly lower than that reported for other ferricytochromes c. On the other hand, the reverse situation applies to the reduction of ferricytochrome cL by reduced methanol dehydrogenase, the reduction occurring instantaneously at pH 7 but much more slowly at pH 9 (ferricytochrome cH was reduced at a 7-fold lower rate, but the rates at pH 7 and 9 were similar). Insignificant reduction was observed with cyclopropanol-inactivated enzyme or with enzyme in the presence of EDTA. In view of the dissimilarities, it is concluded that different mechanisms operate in the autoreduction of ferricytochrome cL and in its reduction by reduced methanol dehydrogenase.     

Measurement of the oxidation-reduction potentials of amicyanin and c-type cytochromes from Paracoccus denitrificans

The oxidation-reduction potentials of four periplasmic electron carrier proteins from Paracoccus denitrificans have been determined. Their midpoint potentials are: amicyanin, 294 +/- 6 mV; cytochrome c-550, 253 +/- 5 mV; cytochrome c-551i, 190 +/- 4 mV; and cytochrome c-553i, 148 +/- 5 mV. Although rapid amicyanin-mediated transfer of electrons from methylamine dehydrogenase to cytochrome c-551i was observed, reduced amicyanin did not reduce oxidized cytochrome c-551i in the absence of methylamine dehydrogenase.     

Complex formation between methylamine dehydrogenase and amicyanin from Paracoccus denitrificans

Two proteins isolated from Paracoccus denitrificans, the copper-containing electron carrier amicyanin and the pyrroloquinoline quinone-containing enzyme methylamine dehydrogenase, have been shown to form a complex. Complex formation between methylamine dehydrogenase and either oxidized or reduced amicyanin resulted in alterations in the absorbance spectrum of the pyrroloquinoline quinone prosthetic group of methylamine dehydrogenase. Binding of amicyanin to the enzyme exhibited positive cooperativity. Complex formation with methylamine dehydrogenase shifted the oxidation-reduction midpoint potential of amicyanin by 73 mV, from +294 to +221 mV, making electron transfer from amicyanin to cytochrome c551 (Em = +190 mV) thermodynamically possible.     

Membrane-bound cytochromes in a sulfate-reducing strict anaerobe Desulfovibrio vulgaris Miyazaki F

Cytoplasmic membranes were isolated from the cells of a sulfate-reducing strict anaerobe Desulfovibrio vulgaris Miyazaki F and membrane-bound cytochromes were characterized. Redox difference spectra at 77 K revealed the presence of cytochromes with the alpha peaks at 552 and 556 nm while CO-binding difference spectra showed the presence of o-type cytochrome(s). Partial purification of the cytochromes demonstrated that the membranes contain cytochromes c550, c551, c556 and possibly d1 besides high molecular mass cytochrome c and cytochrome c3. It turned out that two kinds of novel CO-binding c-type cytochromes are present in the membrane. The membranes and a partially purified fraction showed weak ubiquinol-1 oxidase activity but no cytochrome c oxidase activity. Results suggest that D. vulgaris does not express the heme-copper terminal oxidase under our growth conditions in spite of the presence of the col gene, which is homologous to the gene of subunit I of the aa3-type oxidase.