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.     

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.     

Chemical cross-linking study of complex formation between methylamine dehydrogenase and amicyanin from Paracoccus denitrificans

Two soluble periplasmic redox proteins from Paracoccus denitrificans, the quinoprotein methylamine dehydrogenase and the copper protein amicyanin, form a weakly associated complex that is critical to their physiological function in electron transport [Gray, K. A., Davidson, V. L., & Knaff, D. B. (1988) J. Biol. Chem. 263, 13987-13990]. The specific interactions between methylamine dehydrogenase and amicyanin have been studied by using the water-soluble cross-linking agent 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide (EDC). Treatment of methylamine dehydrogenase alone with EDC caused no intermolecular cross-linking but did cause intramolecular cross-linking of this alpha 2 beta 2 oligomeric enzyme. The primary product that was formed contained one large and one small subunit. Methylamine dehydrogenase and amicyanin were covalently cross-linked in the presence of EDC to form at least two distinct species, which were identified by nondenaturing polyacrylamide gel electrophoresis (PAGE). The formation of these cross-linked species was dependent on ionic strength, and the ionic strength dependence was much greater at pH 6.5 than at pH 7.5. The effects of pH and ionic strength were different for the different cross-linked products. SDS-PAGE and Western blot analysis of these cross-linked species indicated that the primary site of interaction for amicyanin was the large subunit of methylamine dehydrogenase and that this association could be stabilized by hydrophobic interactions. In light of these results a scheme is proposed for the interaction of amicyanin with methylamine dehydrogenase that is consistent with previous data on the physical, kinetic, and redox properties of this complex.     

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.     

Structural comparison of crystal and solution states of the 138 kDa complex of methylamine dehydrogenase and amicyanin from Paracoccus versutus

Methylamine can be used as the sole carbon source of certain methylotrophic bacteria. Methylamine dehydrogenase catalyzes the conversion of methylamine into formaldehyde and donates electrons to the electron transfer protein amicyanin. The crystal structure of the complex of methylamine dehydrogenase and amicyanin from Paracoccus versutus has been determined, and the rate of electron transfer from the tryptophan tryptophylquinone cofactor of methylamine dehydrogenase to the copper ion of amicyanin in solution has been determined. In the presence of monovalent ions, the rate of electron transfer from the methylamine-reduced TTQ is much higher than in their absence. In general, the kinetics are similar to those observed for the system from Paracoccus denitrificans. The complex in solution has been studied using nuclear magnetic resonance. Signals of perdeuterated, (15)N-enriched amicyanin bound to methylamine dehydrogenase are observed. Chemical shift perturbation analysis indicates that the dissociation rate constant is approximately 250 s(-1) and that amicyanin assumes a well-defined position in the complex in solution. The most affected residues are in the interface observed in the crystal structure, whereas smaller chemical shift changes extend to deep inside the protein. These perturbations can be correlated to small differences in the hydrogen bond network observed in the crystal structures of free and bound amicyanin. This study indicates that chemical shift changes can be used as reliable indicators of subtle structural changes even in a complex larger than 100 kDa.     

Redox properties of the quinoprotein methylamine dehydrogenase from paracoccus denitrificans

Paracoccus denitrificans synthesizes a methylamine dehydrogenase that contains a covalently bound form of pyrroloquinoline quinone as a prosthetic group [Husain, M., & Davison, V.L. (1987) J. Bacteriol. 169, 1712-1717]. Anaerobic reductive titration of this enzyme with dithionite proceeded through a semiquinone intermediate with spectral properties quite distinct from those of the oxidized and reduced species. From these data the molar extinction coefficients were calculated at various wavelengths for the three redox states of this enzyme. The semiquinone was slowly reoxidized under aerobic conditions. The fully reduced enzyme was stable in the presence of oxygen and slowly reoxidized by ferricyanide. Reductive titration of methylamine dehydrogenase with methylamine proceeded directly to the fully reduced form of the enzyme without detectable formation of the semiquinone. Electrochemical titrations of the enzyme yielded an overall midpoint potential value for the two-electron couple (fully oxidized/fully reduced) of 100 +/- 4 mV and an n value of 2.15 +/- 0.15.     

pH-dependent semiquinone formation by methylamine dehydrogenase from Paracoccus denitrificans. Evidence for intermolecular electron transfer between quinone cofactors

The quinonoid confactors of Paracoccus denitrificans methylamine dehydrogenase exhibited a pH-dependent redistribution of electrons from the 50% reduced plus 50% oxidized to the 100% semiquinone redox form. This phenomenon was only observed at pH values greater than 7.5. The semiquinone was not readily reduced by addition of methylamine, consistent with the view that this substrate donates two electrons at a time to each cofactor during catalysis. Once formed at pH 9.0, no change in redox state from 100% semiquinone was observed when the pH was shifted to 7.5, suggesting that the requirement of high pH was for formation and not stability of the semiquinone. The rate of semiquinone formation exhibited a first-order dependence on the concentration of methylamine dehydrogenase, indicating that this phenomenon was a bimolecular process involving intermolecular electron transfer between reduced and oxidized cofactors. The rate of semiquinone formation decreased with decreasing ionic strength, suggesting a role for hydrophobic interactions in facilitating electron transfer between methylamine dehydrogenase molecules. Methylamine dehydrogenase was covalently modified with norleucine methyl ester in the presence of 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide (EDC). This modification did not affect the catalytic activity of the enzyme but greatly inhibited the intermolecular redistribution of electrons at high pH. This modification also prevented subsequent cross-linking by EDC of the large subunit of methylamine dehydrogenase to amicyanin, the natural electron acceptor for this enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)     

Microbial metabolism of C1-nitrogen compounds other than cyanide

Abstract The following topics are discussed in this review: the structure of methylamine dehydrogenase and the binding of its pyrrolo-quinoline quinone (PQQ) prosthetic group, the role of the copper protein amicyanin as electron acceptor of the enzyme and the nature of the electron carriers between amicyanin and oxygen in the electron transport chain. Also covered are recent developments in the metabolism of trimethylamine and its N -oxide and N -methylformamides.     

Genetic organization of methylamine utilization genes from Methylobacterium extorquens AM1.

An isolated 5.2-kb fragment of Methylobacterium extorquens AM1 DNA was found to contain a gene cluster involved in methylamine utilization. Analysis of polypeptides synthesized in an Escherichia coli T7 expression system showed that five genes were present. Two of the genes encoded the large and small subunits of methylamine dehydrogenase, and a third encoded amicyanin, the presumed electron acceptor for methylamine dehydrogenase, but the function of the other two genes is not known. The order on the 5.2-kb fragment was found to be large-subunit gene, the two genes of unknown function, small-subunit gene, amicyanin gene. The gene for azurin, another possible electron acceptor in methylamine oxidation, does not appear to be present within this cluster of methylamine utilization genes.     

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.     

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.     

Apparent oxygen-dependent inhibition by superoxide dismutase of the quinoprotein methanol dehydrogenase.

Methanol dehydrogenase activity, when assayed with phenazine ethosulfate (PES) as an electron acceptor, was inhibited by superoxide dismutase (SOD) and by Mn2+ only under aerobic conditions. Catalase, formate, and other divalent cations did not inhibit the enzyme. The enzyme also exhibited significantly higher levels of activity when assayed with PES under anaerobic conditions relative to aerobic conditions. The oxygen- and superoxide-dependent effects on methanol dehydrogenase were not observed when either Wurster's Blue or cytochrome c-55li was used as an electron acceptor. Another quinoprotein, methylamine dehydrogenase, which possesses tryptophan tryptophylquinone (TTQ) rather than pyrroloquinoline quinone (PQQ) as a prosthetic group, was not inhibited by SOD or Mn2+ when assayed with PES as an electron acceptor. Spectroscopic analysis of methanol dehydrogenase provided no evidence for any oxygen- or superoxide-dependent changes in the redox state of the enzyme-bound PQQ cofactor of methanol dehydrogenase. To explain these data, a model is presented in which this cofactor reacts reversibly with oxygen and superoxide, and in which oxygen is able to compete with PES as an electron acceptor for the reduced species.     

Crystal structure of an electron-transfer complex between methylamine dehydrogenase and amicyanin.

The crystal structure of the complex between the quinoprotein methylamine dehydrogenase (MADH) and the type I blue copper protein amicyanin, both from Paracoccus denitrificans, has been determined at 2.5-A resolution using molecular replacement. The search model was MADH from Thiobacillus versutus. The amicyanin could be located in an averaged electron density difference map and the model improved by refinement and model building procedures. Nine beta-strands are observed within the amicyanin molecule. The copper atom is located between three antiparallel strands and is about 2.5 A below the protein surface. The major intermolecular interactions occur between amicyanin and the light subunit of MADH where the interface is largely hydrophobic. The copper atom of amicyanin and the redox cofactor of MADH are about 9.4 A apart. One of the copper ligands, His 95, lies between the two redox centers and may facilitate electron transfer between them.     

Electron transfer in quinoproteins

Soluble quinoprotein dehydrogenases oxidize a wide range of sugar, alcohol, amine, and aldehyde substrates. The physiological electron acceptors for these enzymes are not pyridine nucleotides but are other soluble redox proteins. This makes these enzymes and their electron acceptors excellent systems with which to study mechanisms of long-range interprotein electron transfer reactions. The tryptophan tryptophylquinone (TTQ)-dependent methylamine dehydrogenase (MADH) transfers electrons to a blue copper protein, amicyanin. It has been possible to alter the rate of electron transfer by using different redox forms of MADH, varying reaction conditions, and performing site-directed mutagenesis on these proteins. From kinetic and thermodynamic analyses of the reaction rates, it was possible to determine whether a change in rate is due a change in Delta G(0), electronic coupling, reorganization energy or kinetic mechanism. Examples of each of these cases are discussed in the context of the known crystal structures of the electron transfer protein complexes. The pyrroloquinoline quinone (PQQ)-dependent methanol dehydrogenase transfers electrons to a c-type cytochrome. Kinetic and thermodynamic analyses of this reaction indicated that this electron transfer reaction was conformationally coupled. Quinohemoproteins possess a quinone cofactor as well as one or more c-type hemes within the same protein. The structures of a PQQ-dependent quinohemoprotein alcohol dehydrogenase and a TTQ-dependent quinohemoprotein amine dehydrogenase are described with respect to their roles in intramolecular and intermolecular protein electron transfer reactions.     

Replacement of the axial copper ligand methionine with lysine in amicyanin converts it to a zinc-binding protein that no longer binds copper

The mutation of the axial ligand of the type I copper protein amicyanin from Met to Lys results in a protein that is spectroscopically invisible and redox inactive. M98K amicyanin acts as a competitive inhibitor in the reaction of native amicyanin with methylamine dehydrogenase indicating that the M98K mutation has not affected the affinity for its natural electron donor. The crystal structure of M98K amicyanin reveals that its overall structure is very similar to native amicyanin but that the type I binding site is occupied by zinc. Anomalous difference Fourier maps calculated using the data collected around the absorption edges of copper and zinc confirm the presence of Zn²⁺ at the type I site. The Lys98 NZ donates a hydrogen bond to a well-ordered water molecule at the type I site which enhances the ability of Lys98 to provide a ligand for Zn²⁺. Attempts to reconstitute M98K apoamicyanin with copper resulted in precipitation of the protein. The fact that the M98K mutation generated such a selective zinc-binding protein was surprising as ligation of zinc by Lys is rare and this ligand set is unique for zinc.     

The ''methylamine oxidase'' system of an obligate methylotroph.

The terminal respiratory oxidase was solubilized from membranes of organism 4025, an obligate methylotroph. The partially purified oxidase is probably a cytochrome co. It does not oxidize amicyanin, but it oxidizes 'azurin' and cytochromes cH and cL. By using a complete 'methylamine oxidase' system reconstituted from pure methylamine dehydrogenase, purified oxidase and soluble blue copper proteins and cytochromes, it was confirmed that amicyanin is essential for methylamine oxidation; it could not be replaced by 'azurin' or cytochrome cH or cL. It was shown that the usual mediator between amicyanin and the oxidase is cytochrome cH, with 'azurin' able to replace it during growth at the high copper concentrations required for optimum growth of this unusual methylotroph.     

Cloning, sequencing, and mutation of a gene for azurin in Methylobacillus flagellatum KT.

The gene cluster for methylamine utilization (mau genes) has been cloned from the obligate methylotrophic bacterium Methylobacillus flagellatum KT. Partial sequence data showed that the organization of these genes was similar to that found in Methylophilus methylotrophus W3A1-NS, including the lack of a gene for amicyanin, which had been thought to be the electron acceptor for methylamine dehydrogenase in M. flagellatum KT. However, a gene encoding azurin was discovered at the 3' end of the mau gene cluster, transcribed in the opposite orientation. A mutant with a defect in this gene showed impaired growth on methylamine, suggesting that azurin is involved in methylamine oxidation in M. flagellatum KT.     

A single methionine residue dictates the kinetic mechanism of interprotein electron transfer from methylamine dehydrogenase to amicyanin

Amicyanin is a type 1 copper protein that is the natural electron acceptor for the quinoprotein methylamine dehydrogenase (MADH). A P52G amicyanin mutation increased the Kd for complex formation and caused the normally true electron transfer (ET) reaction from O-quinol MADH to amicyanin to become a gated ET reaction (Ma, J. K., Carrell, C. J., Mathews, F. S., and Davidson, V. L. (2006) Biochemistry 45, 8284-8293). One consequence of the P52G mutation was to reposition the side chain of Met51, which is present at the MADH-amicyanin interface. To examine the precise role of Met51 in this interprotein ET reaction, Met51 was converted to Ala, Lys, and Leu. The Kd for complex formation of M51A amicyanin was unchanged but the experimentally determined electronic coupling increased from 12 cm-1 to 142 cm-1, and the reorganization energy increased from 2.3 to 3.1 eV. The rate and salt dependence of the proton transfer-gated ET reaction from N-quinol MADH to amicyanin is also changed by the M51A mutation. These changes in ET parameters and rates for the reactions with M51A amicyanin were similar to those caused by the P52G mutation and indicated that the ET reaction had become gated by a similar process, most likely a conformational rearrangement of the protein ET complex. The results of the M51K and M51L mutations also have consequences on the kinetic mechanism of regulation of the interprotein ET with effects that are intermediate between what is observed for the reaction of the native amicyanin and M51A amicyanin. These data indicate that the loss of the interactions involving Pro52 were primarily responsible for the change in Kd for P52G amicyanin, while the interactions involving the Met51 side chain are entirely responsible for the change in ET parameters and conversion of the true ET reaction of native amicyanin into a conformationally gated ET reaction.     

Redox properties of quinohemoprotein amine dehydrogenase from Paracoccus denitrificans

Paracoccus denitrificans produces two primary enzymes for the amine oxidation, tryptophan-tryptophylquinone (TTQ)-containing methylamine dehydrogenase (MADH) and quinohemoprotein amine dehydrogenase (QH-AmDH). QH-AmDH has a novel cofactor, cysteine tryptophylquinone (CTQ) and two hemes c. In this work, the redox potentials of three redox centers in QH-AmDH were determined by a mediator-assisted continuous-flow column electrolytic spectroelectrochemical technique. Kinetics of the electron transfer from QH-AmDH to three kinds of metalloproteins, amicyanin, cytochrome c(550), and horse heart cytochrome c were examined on the basis of the theory of mediated-bioelectrocatalysis. All these metalloproteins work as a good electron acceptor of QH-AmDH and donate the electron to the terminal oxidase of P. denitrificans, which was revealed by reconstitution of the respiratory chain. These properties are in marked contrast with those of MADH, which shows high specificity to amicyanin. These electron transfer kinetics are discussed in terms of thermodynamics and structural property.     

Molecular basis for complex formation between methylamine dehydrogenase and amicyanin revealed by inverse mutagenesis of an interprotein salt bridge

Methylamine dehydrogenase (MADH) and amicyanin form a physiologic complex which is required for interprotein electron transfer. The crystal structure of this protein complex is known, and the importance of certain residues on amicyanin in its interaction with MADH has been demonstrated by site-directed mutagenesis. In this study, site-directed mutagenesis of MADH, kinetic data, and thermodynamic analysis are used to probe the molecular basis for stabilization of the protein complex by an interprotein salt bridge between Arg99 of amicyanin and Asp180 of the alpha subunit of MADH. This paper reports the first site-directed mutagenesis of MADH, as well as the construction, heterologous expression, and characterization of a six-His-tagged MADH. alpha Asp180 of MADH was converted to arginine to examine the effect on complex formation with native and mutant amicyanins. This mutation had no effect on the parameters for methylamine oxidation by MADH, but significantly affected its interaction with amicyanin. Of the native and mutant proteins that were studied, their observed order of affinity for each other was as follows: native MADH and native amicyanin > native MADH and R99D amicyanin > alpha D180R MADH and native amicyanin > alpha D180R MADH and R99D amicyanin, and alpha D180R MADH and R99L amicyanin. The alpha D180R mutation also eliminated the ionic strength dependence of the reaction of MADH with amicyanin that is observed with wild-type MADH. Interestingly, the inverse mutation pair of alpha D180R MADH and R99D amicyanin did not restore the favorable salt bridge, but instead disrupted complex formation much more severely than did either individual mutation. These results are explained using molecular modeling and thermodynamic analysis of the kinetic data to correlate the energy contributions of specific stabilizing and destabilizing interactions that are present in the wild-type and mutant complexes. A model is also proposed to describe the sequence of events that leads to stable complex formation between MADH and amicyanin.