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PubMed2002

Genetics of isoprenoid biosynthesis in Paracoccus zeaxanthinifaciens.

The genes coding for all the enzymes involved in the conversion of acetyl-CoA to farnesyl diphosphate (FPP) in the zeaxanthin-producing bacterium Paracoccus zeaxanthinifaciens were cloned and characterized. Two genes encoding enzymes catalysing the condensation of two acetyl-CoA molecules to acetoacetyl-CoA were found. The six enzymes involved in the conversion of acetyl-CoA and acetoacetyl-CoA to isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) are grouped in an operon, designated the mevalonate operon. The gene encoding the enzyme catalysing two consecutive condensations, IPP and DMAPP to geranyl diphosphate (GPP) and IPP and GPP to FPP, is not clustered with any other gene encoding an enzyme of the isoprenoid pathway. Genes encoding enzymes involved in the biosynthesis of poly-hydroxyalkanoate and non-carotenoid isoprenoids found in P. zeaxanthinifaciens are also presented.

Acetyl Coenzyme A↗

PubMed2003

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.

Bacterial Proteins↗

PubMed2000

Binding of O(2) and its reduction are both retarded by replacement of valine 279 by isoleucine in cytochrome c oxidase from Paracoccus denitrificans.

The crystal structure of the heme-copper oxidases suggested a putative channel of oxygen entry into the heme-copper site of O(2) reduction. Changing a conserved valine near this center in cytochrome bo(3) of Escherichia coli to isoleucine caused a significant increase in the apparent K(M) for oxygen with little or no change in V(max), suggesting that oxygen diffusion had been partially blocked [Riistama, S., Puustinen, A., García-Horsman, A., Iwata, S., Michel, H., and Wikström, M. (1996) Biochim. Biophys. Acta 1275, 1-4]. To study this phenotype further using rapid kinetic methods, the corresponding change (V279I) has been made in cytochrome aa(3) from Paracoccus denitrificans. In this mutant, the apparent K(M) for oxygen is 8 times higher than in the wild-type enzyme, whereas V(max) is decreased only to approximately half of the wild-type value. Flow-flash kinetic measurements show that the initial binding of oxygen to the heme of the binuclear site is indeed much slower in the mutant than in the wild-type enzyme. However, the subsequent phases of the reaction with O(2) are also slow although the pure heme-to-heme electron transfer process is essentially unperturbed. It is suggested that the mutation sterically hinders O(2) entry into the binuclear site and that it may also perturb the structure of local water molecules involved in proton transfer to this site.

Amino Acid Substitution↗

PubMed2000

Quantum mechanical interpretation of nitrite reduction by cytochrome cd1 nitrite reductase from Paracoccus pantotrophus.

The reduction of nitrite to nitric oxide in respiratory denitrification is catalyzed by a cytochrome cd(1) nitrite reductase in Paracoccus pantotrophus (formerly known as Thiosphaera pantotropha LMD 92.63). High-resolution structures are available for the fully oxidized [Fülöp, V., Moir, J. W., Ferguson, S. J., and Hajdu, J. (1995) Cell 81, 369-377; Baker, S. C., Saunders, N. F., Willis, A. C., Ferguson, S. J., Hajdu, J., and Fülöp, V. (1997) J. Mol. Biol. 269, 440-455] and fully reduced forms of this enzyme, as well as for various intermediates in its catalytic cycle [Williams, P. A., Fülöp, V., Garman, E. F., Saunders, N. F., Ferguson, S. J., and Hajdu, J. (1997) Nature 389, 406-412]. On the basis of these structures, quantum mechanical techniques (QM), including density functional methods (DFT), were combined with simulated annealing (SA) and molecular mechanics techniques (MM) to calculate the electronic distribution of molecular orbitals in the active site during catalysis. The results show likely trajectories for electrons, protons, substrates, and products in the process of nitrite reduction, and offer an interpretation of the reaction mechanism. The calculations indicate that the redox state of the d(1) heme and charges on two histidines in the active site orchestrate catalysis locally. Binding of nitrite to the reduced iron is followed by proton transfer from His345 and His388 to one of the oxygens of nitrite, creating a water molecule and an [Fe(II)-NO(+)] complex. Valence isomerization within this complex gives [Fe(III)-NO]. The release of NO from the ferric iron is influenced by the protonation state of His345 and His388, and by the orientation of NO on the d(1) heme. Return of Tyr25 to a hydrogen-bonding position between His345 and His388 facilitates product release, but a rebinding of Tyr25 to the oxidized iron may be bypassed in steady-state catalysis.

Binding Sites↗

PubMed2000

Cytochrome cd(1) from Paracoccus pantotrophus exhibits kinetically gated, conformationally dependent, highly cooperative two-electron redox behavior.

Each monomer of the dimeric cytochrome cd(1) nitrite reductase from Paracoccus pantotrophus contains two hemes: one c-type center and one noncovalently bound d(1) center. Potentiometric analysis at 20 degrees C shows substantial cooperativity between the two redox centers in terms of their joint co-reduction (or co-oxidation) at a single apparent potential with an n value of 1.4 +/- 0.1. Reproducible hysteresis is demonstrated in the redox titrations. In a reductive titration both centers titrate with an apparent midpoint potential of +60 +/- 5 mV while in the oxidative titration the apparent potential is +210 +/- 5 mV. However, at 40 degrees C the reductive and oxidative titrations are shifted such that they almost superimpose; each has n = 2. A kinetically gated process that can be correlated with oxidation/reduction-dependent ligand changes at the two heme centers, previously seen by crystallography, is implicated. In contrast, a semi-apoenzyme, lacking the d(1) heme, exhibits a reversible redox titration with a midpoint potential of +242 +/- 5 mV (n = 1). The data with the holoenzyme show how redox changes can themselves generate a gating of the type that is minimally required to account for redox-linked proton pumping by membrane-bound cytochromes.

Allosteric Regulation↗

PubMed2000

The Met99Gln mutant of amicyanin from Paracoccus versutus.

The axial copper ligand methionine has been replaced by a glutamine in the cupredoxin amicyanin from Paracoccus versutus. Dynamic and structural characteristics of the mutant have been studied in detail using UV/Vis, EPR, NMR, cyclic voltammetry, and isomorphous metal replacement. M99Q amicyanin is a blue copper protein with significant spectral and structural similarities to the other cupredoxins umecyanin, stellacyanin, and M121Q azurin. In addition, the functional properties of M99Q amicyanin, as reflected in the electron self-exchange rate constant and midpoint potential (165 mV), have been assessed and compared to values for M121Q azurin. For the latter protein, the published midpoint potential was corrected to the much lower value of 147 mV at pH 7, I = 0.1 M. These values are very similar to the midpoint potential of stellacyanin, which naturally possesses an axial glutamine ligand and has the lowest reduction potential for a naturally occurring cupredoxin. A remarkable feature of M99Q amicyanin, in the reduced state, is the relatively high pK(a) value of 7.1 for its His96 ligand.

Amino Acid Substitution↗

PubMed1993

A new kinetic model for the steady-state reactions of the quinoprotein methanol dehydrogenase from Paracoccus denitrificans.

The reactions of methanol dehydrogenase from Paracoccus denitrificans with artificial electron acceptors, ammonia, cyanide, and substrates have been characterized by steady-state kinetic analysis. Phenazine ethosulfate, a commonly used electron acceptor for this enzyme, was shown to exhibit pronounced substrate inhibition with a K(i) value approximately 20-fold lower than its Km. Wurster's Blue exhibited only relatively mild substrate inhibition and was deemed a more appropriate electron acceptor. Ammonia was an obligatory activator of the enzyme at low concentrations and inhibited a high concentrations. The K(i) value for this inhibition correlated closely with the Kd calculated from a titration of perturbations of the absorption spectrum of methanol dehydrogenase which were caused by the addition of ammonia. Cyanide, which suppressed the peculiar endogenous reaction of methanol dehydrogenase, was also both an activator of substrate-dependent activity and a competitive inhibitor with respect to methanol. Kinetic analysis indicated that the latter two activities corresponded to two distinct binding sites for cyanide. The Ka for cyanide activation correlated closely with the concentration required to inhibit 50% of the endogenous reaction, suggesting that a single binding event is responsible for both of these effects. A model is presented to describe the effects of ammonia and cyanide in the reaction cycle of methanol dehydrogenase, and the physiological relevance of the activation and inhibition by these compounds in vitro is discussed.

Alcohol Oxidoreductases↗

PubMed1993

Resonance Raman spectroscopy of the cytochrome c oxidase from Paracoccus denitrificans.

Resonance Raman spectra are reported for the fully reduced unliganded and cyanide-bound mixed-valence forms of the cytochrome c oxidases from bovine heart and Paracoccus denitrificans in both detergent-solubilized forms and within their natural membrane environments. Comparison of the vibrational patterns observed for these enzymes indicates that overall the heme environments are similar for both. The only major differences seen between the spectra of these two enzymes are for vibrations associated with the low-spin bis(histidine)-coordinated heme cytochrome a. The data reported here serve to further illustrate the close structural and functional relationship between these evolutionarily distant enzymes. However, the data also demonstrate specific differences between the nature of the heme-protein interactions in the cytochrome a binding pocket which may be of mechanistic importance with regard to intramolecular electron transfer in these enzymes.

Animals↗

PubMed1994

Thermodynamic and structural stability of cytochrome c oxidase from Paracoccus denitrificans.

The structural stability of the integral membrane protein cytochrome c oxidase from Paracoccus denitrificans has been measured by high-sensitivity differential scanning calorimetry and Fourier transform infrared spectroscopy. Contrary to the mammalian enzyme or the yeast enzyme, which are composed of 13 subunits, the bacterial enzyme has only three or four subunits, thus providing a unique opportunity to examine the magnitude of the forces that stabilize this enzyme and to establish accurate structural assignments of events observed calorimetrically. In this paper, experiments have been performed with the wild-type enzyme and with a mutant enzyme lacking subunit III. Our results show that subunits I and II form a highly cooperative complex which denatures as a single cooperative unit at 67 degrees C, while subunit III is less stable and denatures 20 degrees C earlier. Reduction of the enzyme causes a large increase in the stability of subunits I and II but has absolutely no effect on subunit III. Despite the lack of a strong interaction between subunit III and the catalytic subunits, the absence of subunit III leads to a turnover-induced loss of electron-transfer activity. The magnitude of the energetic parameters and the infrared spectroscopic experiments indicate that the enzyme does not completely unfold upon thermal denaturation and that significant degrees of structure are preserved. The amount of native alpha-helix structure, which is 45% in the native state, decreases only to 30% after thermal denaturation. Presumably, the residual helical structure existing after thermal denaturation belongs to the intramembranous portions of the protein. The calorimetric behavior of subunit III does not fully conform to that expected for a highly alpha-helical membrane protein. The picture that emerges from these experiments is that, in the temperature-denatured form of the enzyme, most of the extramembranous structural elements are denatured while most of the intramembranous secondary structure is maintained even though native tertiary interactions appear to be disrupted.

Calorimetry, Differential Scanning↗

PubMed2001

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.

Binding Sites↗

PubMed2001

Calcium-dependent conformation of a heme and fingerprint peptide of the diheme cytochrome c peroxidase from Paracoccus pantotrophus.

The structural changes in the heme macrocycle and substituents caused by binding of Ca(2+) to the diheme cytochrome c peroxidase from Paracoccus pantotrophus were clarified by resonance Raman spectroscopy of the inactive fully oxidized form of the enzyme. The changes in the macrocycle vibrational modes are consistent with a Ca(2+)-dependent increase in the out-of-plane distortion of the low-potential heme, the proposed peroxidatic heme. Most of the increase in out-of-plane distortion occurs when the high-affinity site I is occupied, but a small further increase in distortion occurs when site II is also occupied by Ca(2+) or Mg(2+). This increase in the heme distortion explains the red shift in the Soret absorption band that occurs upon Ca(2+) binding. Changes also occur in the low-frequency substituent modes of the heme, indicating that a structural change in the covalently attached fingerprint pentapeptide of the LP heme occurs upon Ca(2+) binding to site I. These structural changes may lead to loss of the sixth ligand at the peroxidatic heme in the semireduced form of the enzyme and activation.

Binding Sites↗

PubMed2001

Electron and proton transfer in the arginine-54-methionine mutant of cytochrome c oxidase from Paracoccus denitrificans.

Arginine 54 in subunit I of cytochrome c oxidase from Paracoccus denitrificans interacts with the formyl group of heme a. Mutation of this arginine to methionine (R54M) dramatically changes the spectral properties of heme a and lowers its midpoint redox potential [Kannt et al. (1999) J. Biol. Chem. 274, 37974-37981; Lee et al. (2000) Biochemistry 39, 2989-2996; Riistama et al. (2000) Biochim. Biophys. Acta 1456, 1-4]. During anaerobic reduction of the mutant enzyme, a small fraction of heme a is reduced first along with heme a(3), while most of heme a is reduced later. This suggests that electron transfer is impaired thermodynamically due to the low redox potential of heme a but that it still takes place from Cu(A) via heme a to the binuclear site as in wild-type enzyme, with no detectable bypass from Cu(A) directly to the binuclear site. Consistent with this, the proton translocation efficiency is unaffected at 1 H(+)/e(-) in the mutant enzyme, although turnover is strongly inhibited. Time-resolved electrometry shows that when the fully reduced enzyme reacts with O(2), the fast phase of membrane potential generation during the P(R )()--> F transition is unaffected by the mutation, whereas the slow phase (F --> O transition) is strongly decelerated. In the 3e(-)-reduced mutant enzyme heme a remains oxidized due to its lowered midpoint potential, whereas Cu(A) and the binuclear site are reduced. In this case the reaction with O(2) proceeds via the P(M) state because transfer of the electron from Cu(A) to the binuclear site is delayed. The single phase of membrane potential generation in the 3e(-)-reduced mutant enzyme, which thus corresponds to the P(M)--> F transition, is decelerated, but its amplitude is comparable to that of the P(R)--> F transition. From this we conclude that the completely (4e(-)) reduced enzyme is fully capable of proton translocation.

Arginine↗

PubMed1984

1H NMR spectroscopy of Paracoccus denitrificans cytochrome c-550.

The 1H NMR spectra of ferri- and ferro-cytochrome c-550 from Paracoccus denitrificans (ATCC 13543) have been investigated at 300 MHz. The ferri-cytochrome c-550 shows hyperfine-shifted heme methyl resonances at 29.90, 29.10, 16.70, and 12.95 ppm and a ligand methionyl methyl resonance at -15.80 ppm (pH 8 and 23 degrees C). Four pH-linked structural transitions were detected in spectra taken as a function of pH. The transitions have been interpreted as loss of the histidine heme ligand (pK less than or equal to 3), ionization of a buried heme propionate (pK = 6.3 +/- 0.2), displacement of the methionine heme ligand by a lysyl amino group (pK congruent to 10.5), and loss of the lysyl ligand (pK greater than or equal to 11.3). The temperature behavior of hyperfine-shifted resonances was determined. Two heme methyl resonances (at 16.70 and 12.95 ppm) showed downfield hyperfine shifts with increasing temperature. The cyanoferricytochrome had methyl resonances at 23.3, 20.1, and 19.4 ppm. NMR spectroscopy did not detect the formation of a complex with azide. The second-order rate constant for electron transfer between ferric and ferrous forms was determined to be 1.6 X 10(4) M-1 s-1. Heme proton resonances were assigned in both oxidation states by cross-saturation and nuclear Overhauser enhancement experiments. Spin-coupling patterns in the aromatic region of the ferro-cytochrome spectrum were investigated.

Cytochrome c Group↗

PubMed1986

Properties of Paracoccus denitrificans amicyanin.

Paracoccus denitrificans synthesizes an inducible, periplasmic, blue copper protein [Husain, M., & Davidson, V.L. (1985) J. Biol. Chem. 260, 14626-14629] that can be classified as an amicyanin on the basis of its ability to accept electrons from methylamine dehydrogenase. The amino acid composition and sequence of the 10 N-terminal residues of this protein have been determined. From these data, it is evident that amicyanin is structurally distinct from azurins as it contains no disulfide bond and an N-terminal sequence that is completely different from the highly conserved N-terminal azurin sequences. Dialysis of reduced amicyanin against potassium cyanide resulted in a nearly quantitative yield of apoamicyanin. Amicyanin and apoamicyanin exhibit fluorescence emission maxima at 314 nm when excited at 280 nm. Addition of 6 M guanidine hydrochloride shifts these emission maxima to 350 nm. The fluorescence intensity of apoamicyanin is 10-fold greater than that of amicyanin. Addition of copper to the apoprotein caused a stoichiometric quenching of fluorescence and restoration of visible absorbance with no concomitant change in absorbance at 280 nm. At least one cysteine residue, which reacts with 5,5'-dithiobis(2-nitrobenzoic acid) in apoamicyanin, does not react in the holoprotein, even in the presence of 6 M guanidine hydrochloride. Reductive and oxidative titrations of amicyanin indicate that it is a one-electron carrier. This amicyanin is also able to accept electrons from the methylamine dehydrogenase isolated from bacterium W3A1, which is taxonomically very different from P. denitrificans.

Amino Acid Sequence↗

PubMed1987

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.

Bacterial Proteins↗

PubMed1988

Preparation of a one-subunit cytochrome oxidase from Paracoccus denitrificans: spectral analysis and enzymatic activity.

Cytochrome c oxidase was isolated from Paracoccus denitrificans as a two-subunit enzyme. Chymotrypsin-catalyzed proteolysis reduced the molecular weight of each subunit by about 8000. The spectral properties of this preparation, as well as its Km for cytochrome c(1.7 muM), remained unchanged with respect to the native enzyme. Vmax was reduced by about 55% when assayed in Triton X-100 or in Triton X-100 supplemented with asolectin. Following further proteolysis by Staphylococcus aureus V8 protease, subunit I remained unchanged as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, whereas subunit II was split into small peptides. These were removed by ion-exchange high-performance liquid chromatography. The one-subunit enzyme had an apparent molecular weight of 43,000. The reduction of molecular weight was also confirmed by the diminution of the ultraviolet/Soret absorption ratio. This value was 1.8-2.1 for the native enzyme and 1.3-1.5 for the one-subunit enzyme. The spectral properties (including the spectrum CO reduced minus reduced) were not modified by the proteolytic treatment, indicating that cytochromes a and a3 were present in equal amounts. The lack of spectral alteration and the known close association of the copper B atom with cytochrome a3 suggest that copper B is also contained within the one-subunit enzyme. The Km of the one-subunit oxidase was similar to that of the two-subunit enzyme; Vmax was decreased by about 50%. The activity of the one-subunit oxidase had a salt-dependent maximum at 30 mM KCl, almost identical with that of the undigested enzyme, and was inhibited by micromolar concentrations of KCN.

Affinity Labels↗

PubMed1990

Spectral and electrochemical properties of glutaryl-CoA dehydrogenase from Paracoccus denitrificans.

Studies of the spectral (UV/vis and resonance Raman) and electrochemical properties of the FAD-containing enzyme glutaryl-CoA dehydrogenase (GCD) from Paracoccus denitrificans reveal that the properties of the oxidized enzyme (GCDox) appear to be invariant from those properties known for other acyl-CoA dehydrogenases such as mammalian general acyl-CoA dehydrogenase (GACD) and butyryl-CoA dehydrogenase (BCD) from Megasphaera elsdenii. However, when either free or complexed GCD is reduced, its spectral and electrochemical behavior differs from that of both GACD and BCD. Free GCD does not stabilize any form of one-electron-reduced GCD, but when GCD is complexed to its inhibitor, aceto-acetyl-CoA, the enzyme stabilizes 20% of the blue neutral radical form of FAD (FADH.) upon reduction. Like GACD, when crotonyl-CoA- (CCoA) bound GCD is reduced, the red anionic form of FAD radical (FAD.-) is stabilized, and excess reduction equivalents are necessary to effect full reduction of the complex. A comproportionation reaction is proposed between fully reduced crotonyl-CoA-bound GCD (GCD2e-CCoA) and GCDox-CCoA to partially explain the stabilization of GCD-bound FAD.- by CCoA. When GCD is reduced by its optimal substrate, glutaryl-CoA, a two-electron reduction is observed with concomitant formation of a long-wavelength charge-transfer band. It is proposed that the ETF specific for GCD abstracts one electron from this charge-transfer species and this is followed by the decarboxylation of the oxidized substrate. At pH 6.4, potential values measured for free GCD and GCD bound to acetoacetyl-CoA are -0.085 and -0.129 V, respectively. Experimental evidence is given for a positive shift in the reduction potential of GCD when the enzyme is bound to a 1:1 mixture of butyryl-CoA and CCoA. However, significant GCD hydratase activity is observed, preventing quantitation of the potential shift.

Chemical Phenomena↗

PubMed1990

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.

Azurin↗