Biochem. J. (1985) 228, 719-726 719Printed in Great Britain

The role of blue copper proteins in the oxidation of methylamine by an obligatemethylotroph

S. Ashley LAWTON and Christopher ANTHONYDepartment of Biochemistry, School of Biochemical and Physiological Sciences, University of Southampton,

Bassett Crescent East, Southampton S09 3TU, U.K.

(Received 10 January 1985/5 March 1985; accepted 18 March 1985)

Organism 4025, an obligate methylotroph, when grown on methylamine in thepresence of a high concentration of copper, contained high concentrations ofmethylamine dehydrogenase and two blue copper proteins, amicyanin and an azurin-type protein; these were purified to homogeneity and characterized. The methyl-amine dehydrogenase is a basic protein (pI8.8) and consists of light and heavysubunits (Mr 14100 and 43000; total Mr 112000). This dehydrogenase differedslightly from other methylamine dehydrogenases in its absorption spectrum and in itslack of thermal stability. Amicyanin, the more abundant blue copper protein, had anMr of 11 500, a midpoint redox potential of 294mV at pH 7.0, and a much lowerisoelectric point (pl 5.3) than other amicyanins. Its absorption maximum was 620nm(7-24nm higher than those of other amicyanins); its absorption coefficient (at620nm) was 3.8 mm-'1 cm-'. The 'azurin' (6% of the blue copper protein) had an Mr of12 500, a midpoint redox potential of 323mV and a high isoelectric point (pl 9.4). Itsabsorption maximum was 620nm, the absorption coefficient (16mM-1 cm-') at thiswavelength being considerably greater than that of any blue copper protein describedpreviously. The partially-purified soluble cytochromes CH and CL were similar to thoseof other methylotrophs. The interactions of the purified redox proteins wereinvestigated in order to elucidate their role in methylamine oxidation. Methylaminedehydrogenase was able to donate electrons only to amicyanin, the rate of reactionbeing 2.04mmol/min per jumol of methylamine dehydrogenase; this is sufficient toaccount for the rate of respiration in whole bacteria. The blue copper proteins wereable to react rapidly with each other and with both the soluble cytochromes c.

Organism 4025 is a white Gram-negative obli-gate methylotroph, unable to grow on methane butable to grow rapidly on methanol or methylamineby way of the ribulose monophosphate pathway(Johanides et al., 1979), and in these features it issimilar to other obligate methylotrophs such asMethylophilus methylotrophus and Methylomonas J(Anthony, 1982, pp. 23-26). An unusual feature oforganism 4025 is its response to copper in thegrowth medium, maximum growth being achievedonly at higher copper concentrations (Vrdoljak etal., 1978). During our preliminary investigations ofthis phenomenon, it was found that organismsgrown on methylamine were blue-green in colour,indicating that they might contain very muchhigher concentrations of blue copper protein thanany other methylotroph previously tested (S. A.Lawton & C. Anthony, unpublished work). It was

assumed that this blue copper protein either wassimilar in function to amicyanin, first described inthe pink facultative methylotroph PseudomonasAMI (Tobari & Harada, 1981), or was similar toazurin, a protein usually associated with electrontransport to nitrite, and also known to be present insome methylotrophs (Tobari, 1984). Amicyaninhas been shown to be the primary electron acceptorfor methylamine dehydrogenase in PseudomonasAMI and Methylomonas J, although it was said tobe absent from trimethylamine-grown Methylo-philus methylotrophus (Burton et al., 1983).The present paper describes an investigation of

the blue copper proteins, present in high concen-trations in organism 4025; these proteins werepurified and characterized, and their interactionswith each other and with cytochromes andmethylamine dehydrogenase are described.

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Methods

Buffer solutionsAll buffer solutions used for preparation of

bacterial extracts and for subsequent purificationof blue copper proteins contained 1.2iM-CUSO4.

Growth ofbacteria andpreparation ofsoluble extractsOrganism 4025 (ZMTF 4025), originally isolated

on methanol by enrichment from soil (Vrdoljak etal., 1977; Johanides et al., 1979), was a gift fromDr. M. Vrdoljak (INA-Razvoj i Istrazivanje,Zagreb, Yugoslavia). Bacteria were grown onmethylamine (0.5%, w/v) or methanol (1%, v/v) inbatch culture on the medium ofAmano et al. (1975)containing 5ml of trace-element solution/l. Thiscontained (g/l): FeSO4,7H,O (0.2); H3BO3(0.003); MnSO4,4H,O (0.02); ZnSO4,7H,O (0.02);Na,MoO4 (0.004); CaCl,,2H,O (0.53);CoCl,,6H,O (0.004). CuSO4,5H,O was addedseparately to give a final concentration in thegrowth medium of 1 mg/l. Growth was in stirredaerated 18-litre batch cultures in 20-litre glassvessels at 30°C. They were harvested by using aSharples Super Centrifuge, washed twice at 40C in20mM-Tris/HCI buffer, pH8.0, and suspended inthe same buffer. Bacteria (lOOg wet wt.) suspendedin 270ml of buffer were disrupted by sonication(three 3min periods), and membranes were re-moved by centrifugation at 380000g for 2h.

Purification of methylamine dehydrogenase, ami-cyanin, 'azurin' and soluble cytochromes c fromorganism 4025 grown on methylamineThe crude soluble extract was passed down a

column (23cmx5cm) of DEAE-cellulose equili-brated with 20mM-Tris/HCl buffer, pH8.0, con-taining 1.2 pM-CUSO4. Methylamine dehydrogen-ase, amicyanin, 'azurin' and cytochrome CH werenot adsorbed, whereas cytochrome CL was elutedby using a linear gradient of 20-300mM-Tris/HClbuffer, pH 8.0. The failure of amicyanin to adsorbon the anion-exchange cellulose is noteworthy,because this protein was later shown to be acidic(pI5.3). The fraction containing methylamine de-hydrogenase, amicyanin, 'azurin' and cytochromeCH was concentrated by ultrafiltration under N2on an Amicon YM2 membrane (Amicon, HighWycombe, Bucks., U.K.) before it was subjected togel filtration on a column (85cm x 4cm) of Sepha-dex G-150 equilibrated with 20mM-Tris/HCl buff-er, pH8.0; this procedure separated the dehydro-genase from amicyanin, 'azurin' and cytochromeCH. The fractions containing methylamine dehy-drogenase were pooled and dialysed against 10mM-potassium phosphate buffer, pH7.0, and appliedto a column (14cm x 5cm) of CM-cellulose equili-brated with the same buffer, and eluted (at 55-

65 mM-phosphate) by using a linear gradient of 10-100mM-potassium phosphate buffer, pH7.0. Thepure dehydrogenase was dialysed against 10mM-Mops/KOH buffer, pH 7.0, and stored at - 17°C.The fractions containing amicyanin, 'azurin' and

cytochrome CH that had been separated from thedehydrogenase by gel filtration were pooled anddialysed against 10mM-Mes/KOH buffer, pH5.5,and concentrated by ultrafiltration under N2 on anAmicon YM2 membrane. This fraction was thensubjected to cation-exchange chromatography on aPharmacia FPLC Mono-S column equilibratedwith lOmM-Mes/KOH buffer, pH 5.5. 'Azurin' andcytochrome CH were adsorbed, and were elutedtogether (at 310mM-NaCl) by using a lineargradient of 0-1.0M-NaCl in 10mM-Mes/KOHbuffer, pH 5.5. The 'azurin' produced by thismethod was about 98% pure, the single contamin-ating protein being cytochrome cH.

Amicyanin, which was not adsorbed on theMono-S column, was dialysed against 10mM-Mops/KOH buffer, pH 7.0, and subjected to anion-exchange chromatography on a Pharmacia Mono-Q column equilibrated with the same buffer.Amicyanin was eluted (at 300mM-NaCl) by using alinear gradient of 0-1.OM-NaCl in 10mM-Mops/KOH buffer, pH 7.0. The pure amicyanin wasdialysed against the same Mops buffer and storedat - 170C.

After the initial anion-exchange chromato-graphy of the crude soluble extracts (above),the cytochrome CL was concentrated by ultrafiltra-tion under N, as described above and dialysedagainst 20mM-Mops/KOH buffer, pH7.0, follow-ed by gel filtration on a Sephadex G-50 column(76cm x 2.5 cm) equilibrated with the same buffer.The pooled fractions containing cytochrome c wereconcentrated and subjected to further gel filtrationon a Sephadex G-150 column (85cm x 4cm)equilibrated with 20mM-Mops/KOH buffer,pH 7.0. The partially pure cytochrome CL wasstored at -17°C.

Purification of 'azurin'ftom organism 4025 grown onmethanolA crude soluble extract was prepared, as

described above, from methanol-grown bacteria;this extract contained no amicyanin. It was thensubjected to anion-exchange chromatography onDEAE-cellulose (15cmx6cm) and gel filtrationon Sephadex G-150 (85cmx4cm) under condi-tions identical with those described for thepurification of amicyanin. Because pure cyto-chrome CH was difficult to purify, the fractionscontaining azurin and cytochrome CH, obtainedfrom Sephadex G- 150 chromatography, were usedfor experiments with the methylamine dehydro-genase. The cytochrome CH preparation contained

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Role of blue copper proteins in oxidation of methylamine by an obligate methylotroph

no dehydrogenases, amicyanin or cytochrome CL;experiments, with pure azurin, showed that methyl-amine dehydrogenase did not reduce azurin. The'azurin' fraction from the gel-filtration column wasthen dialysed against 10mM-potassium phosphatebuffer, pH 6.0, before cation-exchange chromato-graphy on a column (6cm x 6cm) of CM-celluloseequilibrated with the same buffer. 'Azurin' waseluted (at 40-50mM) by using a linear gradient of10-100mM-potassium phosphate buffer, pH6.0.The 'azurin' fraction was then dialysed against10mM-Mes/KOH buffer, pH5.5, and applied to aPharmacia FPLC cation-exchange column (Mono-S) equilibrated with the same buffer. 'Azurin' waseluted (at 750mM-NaCl) by using a linear gradientof 0-1.0M-NaCl in 10mM-Mes/KOH buffer,pH 5.5. The pure protein was dialysed against10mM-Mops/KOH buffer, pH7.0, and stored at-170C.

Polyacrylamide-gel electrophoresisSodium dodecyl sulphate/ 15%-polyacrylamide-

gel electrophoresis was performed as described byLaemmli & Favre (1973) at pH8.3 and the gelswere stained with Coomassie Brilliant Blue (R250)(Weber & Osborn, 1975). To determine theproportion of each protein in samples, peakintegration was performed on gels stained withCoomassie Blue by using a Chromoscan 3 gelscanner (Joyce-Loebl, Gateshead, U.K.). Thismethod showed that the purified methylaminedehydrogenase, amicyanin and 'azurin' consti-tuted more than 96% of the protein present, noother peaks being visible, and the baseline inte-grating to 4% of the total integral. M, values weredetermined by sodium dodecyl sulphate/polyacryl-amide-gel electrophoresis in the Laemmli system(above), with the following proteins as standards:insulin (5700), horse cytochrome c (11 700), lyso-zyme (14000), myoglobin (17200), a-chymotryp-sinogen (24 500), ovalbumin (43000), pyruvatekinase (56000), methanol dehydrogenase (62000)and bovine serum albumin (67000).

Measurement of protein, methylamine dehydrogen-ase, amicyanin, 'azurin' and cytochromes c

Protein was determined by the method ofBradford (1976), with the filtered reagent asdescribed by Spector (1978); crystalline y-globulinwas used as standard. Methylamine dehydrogen-ase was assayed spectrophotometrically with phen-azine methosulphate and 2,6-dichlorophenolindo-phenol as described by Eady & Large (1968), andpolarographically with phenazine methosulphate,as the primary electron acceptor, and 02 at 30°C inan oxygen electrode (Rank Bros., Bottisham,Cambs., U.K.). The reaction contained (in a 2mlvolume) 25mM-Mops/KOH buffer, pH7.5,

0.5mM-phenazine methosulphate and 10mM-methylamine hydrochloride; the reaction wasstarted by addition of enzyme or substrate.

Absorption spectra were recorded as describedpreviously (O'Keeffe & Anthony, 1980), and theconcentrations of blue copper proteins and cyto-chromes c were calculated from spectra by usingthe following wavelength pairs and millimolar ab-sorption coefficients: azurin from Pseudomonasaeruginosa (625nm - 500 nm, 3.5 mM-1 cm-; Brillet al., 1968); 'azurin' from organism 4025(620nm-500nm, 16 mM-1 cm-1); amicyaninfrom organism 4025 (620nm - 500 nm,3.8mmM-lcm-'). Absorption coefficients for thecytochromes c of Pseudomonas AM1 and Methylo-philus methylotrophus were taken from O'Keeffe &Anthony (1980) and Cross & Anthony (1980)respectively. The absorption coefficients for cyto-chromes CH and CL of organism 4025 wereassumed to be the same as those for the Pseudo-monas AMI cytochromes c.

Analytical isoelectric focusingThis was performed with LKB 804-101 Ampho-

line polyacrylamide gels in the pH range 3.5-9.5 asdescribed previously (O'Keeffe & Anthony, 1980).

Amino acid analysis of the blue copper proteinsThis was done by Dr. M. Gore (University of

Southampton) with a Rank-Hilger Chromaspecamino acid analyser after hydrolysis of the proteins(20nmol) with 6M-HCI at 105°C for 24h in sealedevacuated ampoules. Norleucine was used asinternal standard, and cytochrome c and bovineinsulin were used to check recovery of amino acids.

Measurement of midpoint redox potentialsThese were measured at pH 7.0 in 25 mM-

Mops/KOH buffer at 25°C as described by Cross &Anthony (1980). Titration mixtures contained (in atotal- volume of 5.5 ml) 25mM-Mops/KOH buffer,pH 7.0, 100mM-KCl, 100 iM-2,3,5,6-tetramethyl-phenylenediamine, 50 iM-hydroquinone and50 gM-K3Fe(CN)6. Reductive titrations were per-formed with ascorbic acid, and oxidative titrationswere performed with K3Fe(CN)6.

Assessment ofredox proteins as electron acceptorsformethylamine dehydrogenase

Methylamine-dependent reduction by methyl-amine dehydrogenase of cytochromes and bluecopper proteins was measured spectrophotometri-cally at 22°C at the wavelength maxima of theproteins as given above. The reaction mixturescontained (in 1 ml) 25 mM-Mops/KOH buffer,pH 7.0, pure methylamine dehydrogenase (15 nM),electron acceptor (concentration given in Table 3)and 10mM-methylamine (added to start the reac-

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S. A. Lawton and C. Anthony

tions). The specific activity of the dehydrogenaseused in these experiments when measured withphenazine methosulphate as electron acceptor was183 Mmol of dye reduced/min per pmol of dehydro-genase. Pure cytochromes CH and CL from Methylo-philus methylotrophus were prepared as describedby Cross & Anthony (1980) and were a gift fromDr. S. J. Froud (University of Southampton).Azurin from Pseudomonas aeruginosa was a giftfrom Dr. M. Gore (University of Southampton),and horse heart cytochrome c was obtained fromSigma Chemical Co., Kingston-upon-Thames,Surrey, U.K.

Soluble cytochromes c of organism 4025In order to investigate the potential activity of

the soluble cytochromes c as electron acceptorsfrom methylamine dehydrogenase, they were par-tially purified as described in the Methods section.The purified cytochrome CL showed only one cyto-chrome band on sodium dodecyl sulphate/poly-acrylamide-gel electrophoresis and it contained nocopper protein; the cytochrome CH, although freeof amicyanin and cytochrome CL, was contaminat-ed with 'azurin'. The cytochromes CH and CL from

279

Results

Methylamine dehydrogenase of organism 4025The methylamine dehydrogenase was purified

to homogeneity (Table 1), the specific activity ofthe pure protein being similar to that of othermethylamine dehydrogenases tested (Eady &Large, 1968; Matsumoto, 1978; Shirai et al., 1978).The results in Table 1 show that, as in othermethylotrophs, the dehydrogenase constitutedabout 5% of the soluble protein of methylamine-grown bacteria. Its Mr value, as estimated by gelfiltration, was about 112000, and it consisted oftwo types of subunit (Mr 14000 and 43000),suggesting an a2f32 subunit complex similar to thatofother methylamine dehydrogenases (Matsumotoet al., 1978, 1980; Matsumoto & Tobari, 1978). Theisoelectric point (pI8.8) was similar to that ofmethylamine dehydrogenases from other obligatemethylotrophs (Matsumoto, 1978). By contrastwith the enzyme from PseudomonasAM 1, that fromorganism 4025 was not markedly heat-stable, 50%of activity being lost after 7 min at 80°C. As foundfor other methylamine dehydrogenases, its pHoptimum was between pH 7.0 and 7.5.

Fig. 1 shows that the absorption spectrum of thepure dehydrogenase had an absorption maximumat 420nm instead of the usual maximum at 430nm;this presumably reflects a slightly unusual environ-ment of the pyrrolo-quinoline quinone prostheticgroup of this dehydrogenase (see de Beer et al.,1980).

'. 325

I-

420

1AA=0.02

250 300 350 400 450 500 550 600Wavelength (nm)

Fig. 1. Absorption spectra ofpure methylamine dehydro-genase from organism 4025

The dehydrogenase was purified as described in theMethods section; after purification it was in theoxidized form. Spectra were recorded at 22°C in25 mM-Mops buffer, pH 7.0, with the use of a 1Ommlight-path; the concentration of dehydrogenase was0.9 mg/ml. , Spectrum of oxidized dehydrogen-ase;., spectrum of dehydrogenase reduced with10mM-methylamine.

Table 1. Purification of methylamine dehydrogenase from organism 4025The dehydrogenase was assayed in an oxygen electrode with phenazine methosulphate as primary electron acceptor.Details of the assay system and of purification methods are in the Methods section.Purification

step

Crude extractDEAE-celluloseSephadex G-150CM-cellulose

Volume Total activity(ml) (pmol of 02/min)

27040025074

410313282197

Specific activity(pmol of 02/min per mg)

0.0340.1060.3530.666

Yield Purification(0) (fold)

100766948

3.110.419.6

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Role of blue copper proteins in oxidation of methylamine by an obligate methylotroph

organism 4025 were similar in all respects exam-ined to those from other methylotrophs (Anthony,1982, pp 224-229). The cytochrome CH had Mr12 500 and p19.4. The cytochrome CL had M,18000, p13.8 and a midpoint redox potential of284mV.

Amicyanin of organism 4025During growth on methylamine, organism 4025

contained two blue copper proteins, differingmarkedly in their isoelectric points. As shownbelow, methylamine dehydrogenase was specificfor the acidic protein, which was therefore, byanalogy with a similar protein from PseudomonasAMI, called amicyanin (see Tobari & Harada,1981). Synthesis of this protein was induced bygrowth on methylamine, being completely absentduring growth on methanol (S. A. Lawton & C.Anthony, unpublished work).The amicyanin from organism 4025 was purified

to homogeneity as summarized in Table 2. Its Mrwas shown by sodium dodecyl sulphate/polyacryl-amide-gel electrophoresis to be 11 500, and itsisoelectric point (pl) was shown by isoelectricfocusing to be 5.3, which is markedly lower thanthose of amicyanins from Pseudomonas AM1(pI9.3) and Methylomonas J (pI7.7) (Tobari, 1984).Fig. 2 shows that the midpoint redox potential atpH 7.0 was 294mV. Fig. 3 shows the absorptionspectrum of amicyanin, the copper-containingprosthetic group having an absorption maximumat 620nm in the oxidized state, with an absorptioncoefficient of 3.8mm-' cm-1. The complex finestructure of the u.v.-absorption spectrum presum-ably reflects transitions of a limited number oftryptophan and tyrosine residues in individually

distinct environments within the protein, as hasbeen shown for a number of other blue copperproteins (Iwasaki & Matsubara, 1973; Kakutani etal., 1981).

390 r-

370

350

3301-

E 310

290

270

250

'Azurin'

Amicyanin

-1.0 -0.8 -0.6 -0.4 -0.2 0 0.2 0.4 0.6

log Oxidized copper protein\l Reduced copper protein)

0.8 1.0

Fig. 2. Potentiometric titrations oJ amicyanin and 'azurin'Amicyanin and 'azurin' were purified as describedin the Methods section, and titrations were per-formed at 22°C in 25mM-Mops buffer, pH 7.0, asdescribed in the Methods section. Oxidative titra-tions were performed with ferricyanide as oxidant(M); reductive titrations were performed withascorbate as reductant (0). The lines on the Figureare theoretical curves for single-electron transferreactions (n = 1) described in the equation relatingthe measured redox potential (Eh) to the standardmidpoint potential (E0):

Eoxidized protein

Eh = E+59lrog p i~reduced proteinj

Table 2 Purification oJ amicyanin and 'azurin' from organism 4025Details of assay and purification methods are given in the Methods section. 'Azurin' was purified from methanol-grown bacteria, which contained no amicyanin. Amicyanin was purified from methylamine-grown bacteria, whichalso contained some 'azurin'. This was separated from amicyanin at the Mono-S stage, at which the ratio ofamicyanin to 'azurin' was 15: 1. This ratio was assumed to occur in crude extracts for the purposes of calculatingyields and purification factors.

Volume Copper protein(ml) (nmol/mg of protein)

Total copper protein(nmol)

Yield Purification(0) (fold)

AmicyaninCrude extractDEAE-celluloseSephadex G-150Mono-SMono-Q

'Azurin'Crude extractDEAE-celluloseSephadex G-150CM-celluloseMQno-S

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Purificationstep

80831357783

1.536.00

33.976.596.8

54734441306524402334

150100758492

0.0830.912.3717.2681.3

10069564543

10067534132

750500398311292

3.922.250.063.27

10.928.5

208.0978.6

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S. A. Lawton and C. Anthony

620

(a) 'Azurin'

278[ 282

350 450 550 650 750

278268 282

(b) Amicyanin262

290

620

AA = 0.05

250 350 450 550

Wavelength (nm)

I75650 750

Fig. 3. Absorption spectra of blue copper proteins fromorganism 4025

Spectra were recorded at 22°C with the use of a1Omm light-path. Although the blue copper proteinswere in the reduced state in crude soluble fractions,after passage through DEAE-cellulose they were inthe oxidized state. No oxidizing reaction wastherefore necessary before the spectra of oxidizedblue copper protein ( ) were recorded. Reducedproteins were produced by treatment of blue copperproteins with dithionite ( ).

The amino acid composition of amicyanin wasfound to be as follows: Asp (18), Thr (6), Ser (7),Glu (5), Pro (3), Gly (7), Ala (8), Val (9), Met (7),Ile (2), Leu (4), Tyr (1), Phe (11), His (5), Lys (10)and Arg (3). The values in parentheses are thenearest integers. Tryptophan was not determined,but the single tyrosine residue observed in thisamicyanin is consistent with the fine structureobserved in the u.v.-absorption spectrum (seeabove). Although cysteine was not separatelydetermined, it was observed as a peak duringamino acid analysis; this corresponded to at leastone cysteine residue per molecule of amicyanin.The amino acid composition of this amicyanin isclearly different from that of 'azurin' from organ-ism 4025 (see below) and also different from that of

amicyanins and azurins from Pseudomonas AM1and Methylomonas J (Tobari, 1984).

'Azurin' of organism 4025This basic blue copper protein was present

during growth of organism 4025 on methylamineor methanol (S. A. Lawton & C. Anthony,unpublished work). It is similar in many respects toazurin from other bacteria, and so, for conve-nience, and to distinguish it from amicyanin, it isreferred to as azurin-type or 'azurin'. It waspurified to homogeneity as described in Table 2.Extracts of methanol-grown bacteria were used forits preparation in order to avoid complications dueto the amicyanin always present in large amountsduring growth on methylamine. Its isoelectricpoint was high (pI9.4), as found for many otherazurins (Sutherland & Wilkinson, 1963; Iwasaki &Matsubara, 1973; Kakutani et al., 1981; Tobari,1984), notable exceptions being the acidic azurinsfrom Pseudomonas aeruginosa (Fee, 1975) andParacoccus denitrificans (Martinkus et al., 1980).The absorption maximum was 620nm (Fig. 3) andthe absorption coefficient at this wavelength was16mMI cm-1, a value that is considerably greaterthan those for other blue copper proteins (usually2-4mM'Icm- ). This result indicates that theenvironment, and perhaps the ligation, of thecopper atom(s) in this azurin-type protein must beunusual and that it is worthy of further investiga-tion in this respect. The Mr of 'azurin' fromorganism 4025 was 12500 and its midpoint redoxpotential at pH 7.0 was 323mV (Fig. 2).The amino acid composition was found to be as

follows: Asp (15), Thr (8), Ser (5), Glu (8), Pro (1),Gly (6), Ala (9), Val (13), Met (6), Ile (4), Leu (5),Tyr (2), Phe (8), His (7), Lys (16) and Arg (3).Tryptophan was not determined. The values inparentheses are the nearest integers. Althoughcysteine was not separately determined, it wasobserved as a peak during amino acid analysis; thiscorresponded to at least one cysteine residue permolecule of 'azurin'. The amino acid compositionof this 'azurin' is clearly different from that ofamicyanin from organism 4025 (see above) andalso different from the azurins and amicyaninsfrom Pseudomonas AM 1 and Methylomonas J(Tobari, 1984).The 'azurin' described here (from methanol-

grown bacteria) was identical with that partiallypurified from bacteria grown on methylamine withrespect to Mr, pI and absorption spectrum.

Interaction of methylamine dehydrogenase and itspotential electron acceptors

Methylamine dehydrogenase is a quinoproteinhaving as- its prosthetic group pyrrolo-quinolinequinone (de Beer et al., 1980; Anthony, 1982,

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Role of blue copper proteins in oxidation of methylamine by an obligate methylotroph

Table 3. Interactions of methylamine dehydrogenase from organism 4025 with electron-transfer proteinsThe reduction of each potential electron acceptor was measured at 22°C in 25mM-Mops buffer, pH 7.0, containing15 nM-methylamine dehydrogenase, the reaction being started by addition of methylamine (20mM). Details of themethod and materials used are given in the Methods section.

Flectron ascentrir

Isoelectricpoint(nmT

Highestconcentration tested

(inmLL,i"tivu w-- sa%,%,vptvi \e's^, \PsPaJM Lni -lAini FvlL j4jL VIlks -

Amicyanin (organism 4025) 5.3 20.0 2044'Azurin' (organism 4025) 9.4 14.7 0Cytochrome CH (organism 4025) 9.4 10.0 0Cytochrome CL (organism 4025) 3.8 29.8 0Azurin (Pseudomonas aeruginosa) 4.9 32.3 545Cytochrome CH (Pseudomonas AM1) 8.8 27.9 0Cytochrome CL (Pseudomonas AM1) 4.2 8.9 0Cytochrome CH (Methylophilus methylotrophus) 8.9 10.2 0Cytochrome CL (Methylophilus methylotrophus) 4.3 9.9 0Cytochrome c (horse heart) 10.0 250 65

pp 200-206). Although it is usually assayed with anartificial dye (phenazine methosulphate) as elec-tron acceptor, when first described by Eady &Large (1968) it was shown also to be able to reactslowly with mammalian cytochrome c. Tobari &Harada (1981), using methylamine dehydrogenasefrom the pink facultative methylotroph Pseudo-monas AM 1, showed that a blue copper protein(named amicyanin), which was induced duringgrowth on methylamine, was able to act as primaryelectron acceptor. This contrasts with methanoldehydrogenase, also having pyrrolo-quinolinequinone as its prosthetic group, which uses solublecytochrome CL as its electron acceptor. Table 3summarizes the results of an investigation of themethylamine dehydrogenase of organism 4025 (anobligate methylotroph) with respect to thesepotential electron acceptors. It is clear that,although horse heart cytochrome c and azurin fromPseudomonas aeruginosa are able to react withmethylamine dehydrogenase, the only physiologic-al electron acceptor in this organism is the acidicblue copper protein, which we therefore refer tothroughout this work as amicyanin. The Km valuefor amicyanin was 20pm; this is similar to the Kmvalue for amicyanin from Pseudomonas AM1.When a low concentration of amicyanin (0.9 MM)

was introduced into reaction mixtures containingmethylamine dehydrogenase and high concentra-tions of those redox proteins not able to reactdirectly with the dehydrogenase, these proteinsbecame reduced at rates between 30% and 100% ofthe rate measured with amicyanin alone. Thisdemonstrates that, in organism 4025, rapid elec-tron transport can occur from amicyanin to theother blue copper protein ('azurin') and to thesoluble cytochromes c. These cytochromes were

also able to reduce rapidly the oxidized 'azurin'

from organism 4025, the rates of reaction being toogreat to measure in the conventional spectro-photometer.

Discussion

The results described in the present paper showthat, when grown on methylamine, the obligatemethylotroph organism 4025 contains two high-potential blue copper proteins, amicyanin and an

azurin-type protein. The amicyanin constituted94% of the blue copper protein in these methyl-amine-grown bacteria, and was the only redoxprotein from organism 4025 able to act as electronacceptor from methylamine dehydrogenase. Itdiffered from the amicyanin having the samefunction in the pink facultative methylotrophPseudomonas AMI in being an acidic protein,perhaps reflecting the importance of electrostaticinteractions between the acid amicyanin and thebasic dehydrogenase of organism 4025. In Pseudo-monas AMI an acid methylamine dehydrogenase(pI5.2) reacts with a basic amicyanin having a pIof 9.3.

It has been shown (S. A. Lawton & C. Anthony,unpublished work) that the methylamine dehydro-genase, soluble cytochromes c and blue copperproteins are exclusively located in the periplasm oforganism 4025. It can be calculated from Tables 1

and 2 that the dehydrogenase and amicyaninconstitute 5% and 1.6% respectively of the solublecell protein. If it is assumed that the periplasmicvolume is 20% of the total bacterial volume, it canbe shown that the concentrations of methylaminedehydrogenase and amicyanin in the periplasm are180Mm and 580 Mm respectively. The Km of thedehydrogenase for amicyanin is 20 UM, indicatingthat the enzyme is able to act at its maximum rate

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726 S. A. Lawton and C. Anthony

(2.04mmol of amicyanin reduced/min per jimol ofdehydrogenase). Assuming that the soluble cellprotein constitutes 25% of the dry weight of thebacteria, it can be calculated that the rate ofrespiration by way of methylamine dehydrogenaseand amicyanin is 55.4nmol of 02 consumed/minper mg dry wt. of bacteria. This is similar to themeasured rate of respiration, by whole bacteria,with methylamine as substrate, which was usuallybetween 100 and 150nmol of 0, consumed/minper mg dry wt. Although such calculations cannever be accurate, because they are based neces-sarily on many assumptions, this result does clearlydemonstrate that the reaction between methyl-amine dehydrogenase and amicyanin is likely tobe of physiological importance. This conclusion issupported by the observation that amicyanin isinduced during growth on methylamine and thatno other redox protein from organism 4025 wasable to interact with methylamine dehydrogenase.The results above demonstrated that amicyanin

is able to donate electrons to the second bluecopper protein, 'azurin', and to the soluble cyto-chromes CH and CL, and also that these proteinsare able to react rapidly with each other. Thus,although amicyanin has been shown to be theunique electron acceptor from methylamine dehy-drogenase, the route for electron transport betweenamicyanin and the cytochrome oxidase (cyto-chrome o) is not yet certain.

It should be noted that, although the presentstudy clearly shows that the blue copper protein insuch abundance in organism 4025 is mainlyamicyanin, and that this is probably the onlyelectron acceptor from methylamine dehydrogen-ase in this organism, no conclusion can be drawnwith respect to the reason for the high concentra-tions of amicyanin produced in this organismcompared with Pseudomonas AM1.The demonstration that two obligate methylo-

trophs (organism 4025 and Methylomonas J) useamicyanin as electron acceptor from methylaminedehydrogenase suggests that the failure to observeblue copper proteins in the obligate methylotrophMethylophilus methylotrophus when grown on tri-methylamine (Burton et al., 1983) might be due tothe difficulty of observing blue copper proteins inthe presence of high concentrations of cytochromec, and not due to an alternative system for electrontransport from methylamine in that organism.

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