Biochem. J. (1980) 192,411-419Printed in Great Britain

411

The two cytochromes c in the facultative methylotroph Pseudomonas AM 1

David T. O'KEEFFE and Christopher ANTHONYDepartment ofBiochemistry, University ofSouthampton, Southampton S09 3TU, U.K.

(Received 1I February 1980/Accepted 18 July 1980)

It was previously suggested that there is only one soluble cytochrome c in PseudomonasAMI, having a molecular weight of 20000, a redox midpoint potential of about+260mV and a low isoelectric pint [Anthony (1975) Biochem. J. 146, 289-298;Widdowson & Anthony (1975) Biochem. J. 152, 349-3561. A more thorough examina-tion of the soluble fraction of methanol-grown Pseudomonas AM 1 has now revealed thepresence of two different cytochromes c. These were both purified to homogeneity byacid treatment, ion-exchange chromatography, gel filtration, chromatography onhydroxyapatite and preparative isoelectric focusing. Molecular weights were determinedby sodium dodecyl sulphate/polyacrylamide-gel electrophoresis; midpoint redox poten-tials were determined directly by using platinum and calomel electrodes; isoelectricpoints were estimated by electrophoresis and by the behaviour of the two cytochromeson ion-exchange celluloses. The more abundant cytochrome CH (A.ax. 550.5 nm) had alow molecular weight (11 000), a midpoint potential of about +294mV and a high iso-electric point, not being adsorbed on DEAE-cellulose in 20mM-Tris/HCl buffer, pH 8.0.The less abundant cytochrome CL ('max. 549nm) was about 30% of the total; it had ahigh molecular weight (20900), a midpoint potential of about +256mV and a low iso-electric point, binding strongly to DEAE-cellulose in 20mM-Tris/HCI buffer, pH8.0.The pH-dependence of the midpoint redox potentials of the two cytochromes c werevery similar. There were four ionizations affecting the redox potentials in the pH rangestudied (pH 4.0-9.5), two in the oxidized form (pK values about 3.5 and 5.5) and twoin the reduced form (pK values about 4.5 and 6.5), suggesting that the ionizing groupsinvolved may be the two propionate side chains of the haem. Neither of the cytochromesc was present in mutant PCT76, which was unable to oxidize or grow on C I

compounds, although still able to grow well on multicarbon compounds such assuccinate. Whether or not these two cytochromes c have separate physiologicalfunctions is not yet certain.

Pseudomonas AM 1 is a facultative methylotrophunable to grow on methane but able to grow on

other C 1 compounds and on a wide range ofmulticarbon compounds such as succinate. Electrontransport from methanol requires an unusualmethanol dehydrogenase and cytochrome c, but notcytochrome b (Anthony, 1975; Widdowson &Anthony, 1975; Netrusov & Anthony, 1979).Cytochrome c is also involved in methanol oxidationin methanotrophs (Higgins, 1979) and in Para-coccus denitrificans (van Verseveld & Stouthamer,1978; Bamforth & Quayle, 1978). Whether or notmethanol dehydrogenase and cytochrome c interactdirectly is not yet certain (see Anthony, 1975; Duineet al., 1979). In order to investigate the possibility of

Abbreviation used: SDS, sodium dodecyl sulphate.

Vol. 192

such a direct reaction we set out to purify andcharacterize the cytochrome c of PseudomonasAM 1. During this work it was found that thismethylotroph produces two species of cytochrome c,and that the description of cytochrome c previouslypublished from our laboratory (Anthony, 1975) w-asin fact a composite, and thus erroneous, descriptionof two different cytochromes c. The present paperdescribes the complete purification and characteri-zation of these two different species of cytQchrome cfrom Pseudomonas AM 1.

It should be noted that the cytochromes c frommethylotrophs are unusual in binding CO to someextent; they are therefore sometimes referred to.ascytochromes cco. Because we consider that thisphenomenon has not been sufficiently investigatedand because its physiological significance (if any)

0306-3283/80/1104 11-09$01.50/1 (© 1980 The Biochemical Society

D. T. O'Keeffe and C. Anthony

has not yet been elucidated, we have avoided thisterminology.

In the present paper the subscripts H and L areused to denote cytochrome c with high and lowisoelectric points respectively. Numerical subscriptshave been avoided in order to prevent confusion withmitochondrial cytochrome cl, cytochrome c2 ofphotosynthetic bacteria, cytochromes C4 and C5 ofAzotobacter etc.A preliminary report of this work has been

published (O'Keeffe & Anthony, 1979).

Materials and methods

ChemicalsAll chemicals were obtained from BDH

Chemicals, Poole, Dorset, U.K., except the follow-ing: 2,3,5,6-tetramethyl-p-phenylenediamine (fromAldrich Chemical Co., Gillingham, Dorset, U.K.);acrylamide (from Koch-Light Laboratories, Coln-brook, Bucks., U.K.); Sephadex G-150, G-75, G-50and G-25 (from Pharmacia Fine Chemicals,Uppsala, Sweden); 4-morpholinepropanesulphonicacid, Tris, insulin, myoglobin, ovalbumin and trypsinfall from Sigma (London) Chemical Co., Kingstonupon Thames, Surrey, U.K.]; DEAE-cellulose andCM-cellulose (from Whatman, Maidstone, Kent,U.K.); hydroxyapatite (from Bio-Rad Laboratories,Richmond, CA, U.S.A.).

Organisms and growth mediaPseudomonas AM 1 (N.C.I.B. 9133) was obtained

from the National Collection of Industrial Bacteria,Torry Research Station, Aberdeen, Scotland, U.K.Stock cultures were maintained on methylamine/agar. The defined liquid growth medium of Mac-Lennan et al. (1971) was used, and methanol(0.4%, w/v) was used as carbon source.

Growth and harvesting ofbacteriaBacteria were grown as stirred aerated 18-litre

batch cultures in 20-litre glass pots at 300C. Thebacterial cultures were harvested at 40C by using aSharples Super centrifuge. They were washed twicein 20mM-Tris/HCl buffer pH 8.0, at 40C beforedisruption.

Measurement ofabsorption spectraAll spectra were recorded with a Cary 118C

double-beam spectrophotometer (Varian Associates,Walton on Thames, Surrey, U.K.). Unless otherwisestated, cuvettes of 10mm light-path were used.Spectra were recorded at 20-250C unless otherwisestated. The scan speed was usually 1 nm/s and thepen period was Is. The slit-width was 0.03 mm,which corresponds to a spectral bandwidth at550nm of 0.6nm. That maximum resolution wasbeing achieved was confirmed by scanning at slower

speeds (0.2nm/s) with narrower slits (0.02mm). Thereductant was dithionite and the oxidant wasferricyanide for reduced-minus-oxidized differencespectra. Absolute spectra were obtained by measur-ing the spectra of dithionite- or ascorbate-reducedcytochrome against a reference solution containingthe same buffer as the cytochrome solution. Formeasurement of (reduced-plus-CO)-minus-reduceddifference spectra, cytochrome in both cuvettes wasreduced with solid Na2S204 followed by bubblingwith CO through one of the cuvettes for 2min, andthe difference spectra were recorded immediatelyand at intervals up to 60min. The cuvettes weresealed with SubaSeal caps to exclude air duringincubations with CO. Base-lines were alwayschecked before recording of spectra. Spectra at77K were obtained by placing Perspex [poly(methylmethacrylate)] cuvettes containing cytochrome sam-ples in liquid N2 and then quickly transferring themto an insulated liquid-N2-containing box, placed inthe sample position of the spectrophotometer. Thecuvettes (2mm light-path) were made by separatingtwo sheets of Perspex, forming the optical surfaces,by an aluminium 'former', which also served as aheat conductor. The (A550-A575)/A28o ratios werecalculated by using untreated (oxidized) cytochromefor the A280 measurement and the dithionite-reducedcytochrome for the A 550-A575 measurement(Ambler, 1963).

Purification ofcytochromes cH and cLThese methods are adapted from those pre-

viously described (Anthony, 1975). Bacterial cells(200g wet wt.) were suspended in 150ml of20mM-Tris/HCl buffer, pH 8.0, and ruptured in aFrench pressure cell (American Instrument Co.,Silver Spring, MD, U.S.A.) at 1OOMPa followed bysonication at 20C for a total of 9min. Whole cellsand cell debris were removed by centrifugation at40000g for 2h at 40C. The resultant pellet wassubjected to a second sonication and high-speedcentrifugation, and the two high-speed supernatantswere combined.The pH of the high-speed supernatant was

lowered to pH4.0 with 1 M-HCl, and the resultantprecipitate was removed by centrifugation. Thesupernatant was adjusted to pH 7.0 with 1 M-NaOHand then dialysed in a beaker dialyser (Biofibre 50;Bio-Rad Laboratories) against 4 litres of 20mM-Tris/HCl buffer, pH 8.0, before application to aDEAE-cellulose column (26cm x 3.5cm) equilib-rated with the same buffer.

Cytochrome cH and methanol dehydrogenase didnot bind to the DEAE-cellulose. The eluate con-taining the cytochrome and dehydrogenase wasconcentrated by ultrafiltration on an Amicon PM10membrane (Amicon, High Wycombe, Bucks., U.K.)under N2 before gel filtration on a Sephadex G-150

1980

412

Two cytochromes c of Pseudomonas AM 1

column (85 cm x 4 cm) equilibrated with a mixture of100mM-Tris/HCI buffer, pH 8.0, and 200mM-NaCl.After gel filtration cytochrome CH was dialysedagainst 10mM-sodium phosphate buffer, pH 6.0, andthen subjected to chromatography on a CM-cellulose column (24cm x 4cm) equilibrated with10mM-phosphate buffer, pH 6.0. The cytochromewas eluted at 40-45-mM-sodium phosphate when alinear gradient of 10-150mM-sodium phosphatebuffer, pH 6.0, was applied to the column in a totalvolume of 250ml. The eluted cytochrome was againconcentrated by ultrafiltration before gel filtration ona Sephadex G-50 column (93 cmx 1.8cm) in1OOmM-Tris/HCl buffer, pH 8.0. The cytochromewas then dialysed against 10mM-sodium phosphatebuffer, pH 6.0, before chromatography and step-wise elution from a hydroxyapatite column(1.5 cm x 2.8 cm). The cytochrome was subse-quently extensively dialysed against distilled waterand stored at-17°C.

Cytochrome CL was eluted from the DEAE-cellulose column in 220mM-Tris/HCI when a lineargradient of 100-300mM-Tris/HCI buffer, pH 8.0,was applied in a total gradient volume of 2 litres. Thecytochrome was then concentrated by ultrafiltrationand applied to a Sephadex G-50 column. The pooledcytochrome fractions were again concentrated byultrafiltration and then applied to a Sephadex G- 150column. The resultant cytochrome was extensivelydialysed against distilled water.

Although both cytochromes at this stage ran assingle bands during SDS/polyacrylamide-gel electro-phoresis, isoelectric focusing showed one very minorcontaminant in cytochrome CH and several proteinbands in cytochrome CL. The cytochromes weretherefore subjected to preparative isoelectricfocusing.

Electrofocusing ofcytochromes CH and CLPreparative isoelectric focusing of the cyto-

chromes was performed in a Sephadex G-50 gel byusing the LKB2117 Multiphor system. CytochromeCH was electrophoresed in the pH 8-9.5 range at aninitial voltage of 0.39kV at 20.5 mA. Cytochrome CLwas electrophoresed in the pH range 3.5-5.5 at aninitial voltage of 0.5 kV at 16 mA. After electro-phoresis for 15 h at 10°C at a limiting power of 8Wthe gel was sectioned and the pH of each section wasmeasured directly with a pH electrode. The cyto-chromes were easily visible as coloured bands andwere eluted from the granular gel with distilledwater. Ampholines used to create the pH gradientwere removed by gel filtration on Sephadex G-50.

Analytical electrofocusing was done on LKB804-101 Ampholine polyacrylamide-gel plates in thepH 3.5-9.5 range in the LKB 2117 MultiphorUniversal apparatus. Before staining, gels were fixedin a mixture of trichloroacetic acid and sulpho-

salicylic acid (57.5 and 17.25 g respectively in500ml of water). Protein bands were stained withCoomassie Brilliant Blue (0.46 g in 400ml ofdestaining solution). Destaining solution was anequal mixture of ethanol (25%, v/v) and acetic acid(8%, v/v).SDS/polyacrylamide-gel electrophoresis

SDS/polyacrylamide-gel electrophoresis wasperformed by the method of Swank & Munkres(1971) with polyacrylamide gels (12.5% acryl-amide) containing 6M-urea at pH6.8. For esti-mation of molecular weight the following proteinstandards were used (mol.wts. in parentheses):insulin (5700), horse heart cytochrome c (1 1 700),lysozyme (14 100), myoglobin (17200), trypsin(23 000) and ovalbumin (42000). Proteins werestained with Coomassie Brilliant Blue (see above).

Determination ofhaemThe haem content of the cytochromes was

determined by the method of Rieske (1967) by usinga mixture containing equal volumes of pyridine and0.5 M-NaOH. The concentration of haem wascalculated from the difference spectrum of thereduced and oxidized pyridine haemochromeby assuming an absorption coefficient of19.1 m l * cm-' for the 550nm-minus-575nmwavelength pair.

Measurement ofmidpoint redox potentialsMidpoint redox potentials were measured by the

method of Dutton et al. (1970) by using platinumelectrodes with a salt bridge completing the circuit toa calomel electrode. The reaction mixture con-tained 25 mM-buffer, 100mM-KCl, 50M-2,3,5,6-tetramethylphenylenediamine and 4.3-8.6puM-cyto-chrome c. Titrations were performed by injection ofmicrolitre quantities of K3Fe(CN)6 (which alsoacted as mediator) or sodium ascorbate through sidearms in the redox cells. The reaction mixture wasconstantly stirred, and the extent of reduction of thecytochromes was determined by recording thespectrum between 535 and 575nm at approx. 5mVsteps in potential. The difference in potentialbetween the platinum and reference electrodes wasmeasured with a digital voltmeter and a correctionmade to give the potential with respect to thehydrogen half-cell (an addition of 247 mV). Alltitrations were performed under 02-free N2. Residualtraces of 02 present in commercial 'white spot' N2were removed by passing the gas through an'02-scrubbing' solution as described by Sweetser(1967).The following buffer solutions were used: acetic

acid/sodium acetate (pH 4.0-5.8); NaH2PO4/NaOH (pH 5.8-6.5); Mops (4-morpholinepropane-sulphonic acid) (pH 6.5-7.9); Hepes [4-(2-hydroxy-

Vol. 192

413

D. T. O'Keeffe and C. Anthony

ethyl-l-piperazine-ethanesulphonic acid] (pH6.8-8.2); Tris (pH 7.5-8.8); 2-(N-cyclohexylamine)-ethanesulphonic acid (pH9.0-10.1); cyclohexyl-aminepropanesulphonic acid (pH 9.7-1 1.1).

Results

Purification ofcytochromes CH and cLTable 1 summarizes the purification procedure

used for the two soluble cytochromes c of Pseudo-monas AM 1. They were initially separated bychromatography on DEAE-cellulose at pH 8.0.Cytochrome CH is the fraction having a highisoelectric point and thus unable to bind to DEAE-cellulose. Cytochrome CL is the fraction binding toDEAE-cellulose (the subscript L indicating lowisoelectric point). These subscripts are used becausethe a-band maxima are only 1.5 nm apart and,although reproducible, the cytochromes are noteasily identifiable by wavelength maxima alone.

The results in Table 1 indicate that the molar ratioof cytochrome CH to cytochrome CL was about2.6: 1. When extract was passed through DEAE-cellulose without prior acid treatment, the propor-tion of cytochrome CH to CL was the same. Likewisethe same proportion of the two cytochromes wasdemonstrated in acid-treated extracts of succinate-grown cells.

Isoelectric points ofcytochromes CH and CLBefore the final stage of purification (preparative

electrophoresis) both cytochromes c appearedhomogeneous on SDS/polyacrylamide-gel electro-phoresis. However, analytical electrofocusing onpolyacrylamide-gel plates as described in theMaterials and methods section showed a lack ofhomogeneity in the preparations, and so a finalpurification stage of electrofocusing was included.This separated a non-cytochrome impurity (less than2% of the total protein) from both cytochromes c.After preparative isoelectric focusing, cytochromeCH was eluted as a single cytochrome band of iso-electric point 8.8. Cytochrome CL consisted of fourcytochrome bands; the main component (90% ofcytochrome CL) had an isoelectric point of 4.2, andthe three minor components had isoelectric points of3.9 (1% of cytochrome CL), 4.0 (4% of cytochromeCL) and 4.3 (5% of cytochrome CL). All propertiesdescribed in the present paper relate to the majorcomponent of cytochrome CL.

Spectral characteristics of cytochromes CH and CLThe two cytochromes c differed by 1.5 nm in their

absorbance maxima and they had slightly differentmolar absorption coefficients (Fig. 1 and Table 2).The (A-AA575)/A280 ratios were 1.13 and 1.0 for

cytochromes CH and CL respectively; these values aresimilar to those obtained for species of cytochrome c

A(

Table 1. Purification oftwo cytochromes cfrom methanol-grown Pseudomonas AMIDetails of methods are given in the Materials and methods section. Cytochrome CH is the cytochrome having a highisoelectric point (thus not binding to DEAE-cellulose). Cytochrome CL has a low isoelectric point and so binds toDEAE-cellulose. Before separation on DEAE-cellulose, the cytochrome concentrations were calculated by usingan average molar absorption coefficient of 28.5 mm-' cm-'. After separation, the coefficients were 26 for cyto-chrome CL and 31 for cytochrome CH. Because accurate cytochrome determinations are impossible in the crudeextract, the yield and purification factors for later purification stages were calculated from the amount and specificactivity in the supematant after acid treatment. This initial treatment yielded a purification of about 3-fold in about100% yield. The final electrophoretic treatment of cytochrome CL produced one major band plus four minor bands;these were measured separately and the total amounts were combined to give the value in the Table.

Total Total Concn. ofVolume protein cytochrome cytochrome Yield Purification

urification step (ml) (mg) (fmol) (nmol/mg) (%) factorcid treatment 370 1655 6.0 3.6 100 1

Cytochrome c,DEAE-celluloseSephadex G-150CM-celluloseSephadex G-50HydroxyapatiteElectrofocusing

Cytochrome CLDEAE-celluloseSephadex G-50Sephadex G-150Electrofocusing

590140200120565.2

1628010210.4

59044822637.79.339.2

79048.417.313.4

3.683.523.22.531.171.15

1.420.740.670.64

6.27.914.267.1125125

1.7915.33948

615853421919

19121111

1.72.23.9

10.83535

0.54.3

1113

1980

414

Two cytochromes c ofPseudomonas AM 1

containing two residues of tryptophan per moleculeof protein (Ambler, 1963; Gurtler & Horstman,1970). The ratio for cytochromes with only one

416.5 550.5

410

Iz I 521.5

200 250 300 350 400 450 500 550 600

416(b) I° 549

410

520

200 250 300 350 400 450 500 550 600Wavelength (nm)

Fig. 1. Absorption spectra ofcytochromes c, and CL(a) Cytochrome CH (1.2pM); (b) cytochrome CL(1.46,UM). Continuous lines are spectra of cyto-chrome reduced with dithionite; broken lines arespectra of untreated (oxidized) cytochrome.

tryptophan residue per molecule is usually morethan 2.The a-band absorbance maxima of both byto-

chromes were lowered to about 548 nm at 77K (Fig.2); there was a 5-7.5-fold intensification of thisband, but no splitting was observed. In this respectthese cytochromes differ from most other solublecytochromes c, in which splitting of the a-band atlow temperatures is usual (Lemberg & Barrett,1973), including two cytochromes c from theobligate methylotroph Methylophilus methylo-trophus (Cross & Anthony, 1980) and the solublecytochrome c found in supernatant fractions of themethanol-grown Pseudomonas extorquens (Tonge etal., 1974).

Treatment with alkaline pyridine gave typicalpyridine haemochrome spectra for the two cyto-chromes with absorbance maxima at 550nmin dithionite-reduced - minus - ferricyanide-oxidizeddifference spectra. Assuming the molecular weightsmeasured below and an absorption coefficient of19.1 mm-' cm-' (A550-A575) (Rieske, 1967), thenthe haem/protein ratios were 0.79 and 0.97 forcytochromes CH and CL respectively, indicating thatthese cytochromes are similar to most other bac-terial cytochromes c with high midpoint potentials inhaving one haem group per molecule of protein.

Molecular weights ofcytochromes CH and CLThe molecular weights of cytochromes CH and CL

were 11000 and 20 900 respectively when measuredby SDS/polyacrylamide-gel electrophoresis. Similarmolecular weights were obtained by gel filtration onSephadex G-50 and G-75, indicating that both of thecytochromes were monomers. That SDS/polyacryl-amide-gel electrophoresis is able to denature dimericcytochrome c has been shown for Azotobactervinelandii cytochrome c5 by Swank & Burris (1969).

Table 2. Properties ofthe two soluble cytochromes c ofPseudomonas AM]

Relative proportions in crude extractsIsoelectric pointMolecular weightMidpoint redox potential at pH 7.0Absorption maxima of ferrocytochrome (a, y)Absorption coefficients (mM- cm-') (a, y)Absorption maximum of ferricytochrome (y)Ratio of a-absorption/y-absorption (ferrocytochrome)Absorption maxima of difference spectrum (a(, y)Absorption coefficients (mM-' * cm-') ((, y)Absorption maxima at 77KRatio of (Aa-A575)/A28o at 200C

Haem groups/molecule of protein% CO-bindingAutoxidizabilityReduction by methanol dehydrogenase (see O'Keeffe & Anthony, 1980)

Vol. 192

Cytochrome CH72%8.811000+294mV550.5 nm, 416.5 nm31, 162410nm5.23550nm, 416nm22.5, 77.35548nm1.13

36%SlowSlight

Cytochrome CL28%4.220900+256mV549nm, 416nm26, 163410nm6.25549nm, 416nm21.8, 64.5548nm1.0

72%SlowExtensive

415

D. T. O'Keeffe and C. Anthony

(a) Il

520

526.5

547.25

417

(b) Ln In

518.5

526.5

400 420 440 490 510 530 550 570Wavelength (nm)

Fig. 2. Reduced-minus-oxidized difference spectra ofcytochromes cN and cL at 77K

(a) Cytochrome cH (4puM); (b) cytochrome c(5.2,UM). Cytochromes were dissolved in 30% (v/vIglycerol and 2mm-light-path cuvettes were used (seethe Materials and methods section).

(a)

11

350 400 450 500 550 600

(b)

I

549

Autoreduction ofcytochromes cH and cLBoth pure cytochromes c were shown to be

reduced by methanol dehydrogenase in the absenceor presence of methanol (see Anthony, 1975). Thesignificance of this in relation to the function of thecytochrome c in methylotrophic bacteria was madeeven more difficult to interpret by the observationthat at pH 9.5 both of the cytochromes becamerapidly reduced in the absence of added reductant orof methanol dehydrogenase. This autoreduction andthe reduction by methanol dehydrogenase are dis-cussed in detail elsewhere (O'Keeffe & Anthony,1980).

Autoxidizability of the cytochromes and their reac-tion with CO

Neither cytochrome was rapidly autoxidizable.During the separation procedures, after removal ofmethanol dehydrogenase both cytochromes becameslowly oxidized; whenever methanol dehydrogenasewas present the cytochromes were fully reduced.After reduction with the minimum volume of dilute

350 400 450 500 550 600Wavelength (nm)

Fig. 3. Reaction ofcytochromes cH and cL with CO(a) Cytochrome cH (3.5pM); (b) cytochrome CL(3.8,M). , Reduced cytochrome c; -------reduced cytochrome c after 30 min exposure to CO.The intermediate spectrum (----) was drawn after7 min exposure to CO.

dithionite, the time taken for 50%o oxidation of thepure cytochromes was 15min and 8min for cyto-chromes cH and cL respectively. These results arguestrongly against the possibility of an oxidasefunction for these cytochromes.The reaction of CO with cytochrome c of bacteria

capable of oxidizing methane has led to the proposalthat this cytochrome may have an oxygenase or

1980

416

Two cytochromes c ofPseudomonas AM 1

oxidase function, and this has been extended topropose an oxidase function for cytochrome c in thefacultative methylotroph Pseudomonas extorquens,which does not grow on methane and is similar inmost respects to Pseudomonas AM1 (Tonge et al.,1975, 1977). In spite of this important conclusionwith respect to reaction with CO, no full spectra ofthe CO-ferrocytochrome complex have been pub-lished. Such spectra for the pure cytochromes c fromPseudomonas AM 1 are presented in Fig. 3 and(reduced + CO)-minus-reduced difference spectraare shown in Fig. 4. The fact that the Soretabsorption maximum of cytochrome CL (412nm)was the same wavelength as the Soret maximumfor the (reduced + CO)-minus-reduced differencespectrum indicates that a high proportion of thecytochrome CL was bound to CO. By contrast, therelatively slight shift in the Soret maximum ofcytochrome CH after reaction with CO indicates thatrelatively less of the cytochrome c was bound to CO.The simplest interpretation of the spectra presentedhere is the same as previously published (Widdow-son & Anthony, 1975); both cytochromes c appearto form complexes with CO having absorptionmaxima at 412 nm, the dissociation constant ofcytochrome CH being considerably higher than thatof cytochrome CL. The rate of reaction of CO withcytochrome CH was about half that with cytochromeCL. About 50% of the cytochrome CL that wouldfinally bind had done so after 5min reaction withCO.

For purposes of comparison it is convenient toestimate the percentage of cytochrome c binding toCO in CO-saturated solution, and for this purpose amolar absorption coefficient must be assumed.Bartsch & Kamen (1960) calculated a molarabsorption coefficient of 165 mm-' - cm-' for the'peak-minus-trough' values in the (reduced + CO)-minus-reduced difference spectrum of Chromatiumcytochrome c, which contains three haem groups permolecule. This was used by Weston & Knowles(1974) and by Tonge et al. (1974, 1975, 1977) forcalculation of the percentage of cytochrome cbinding CO in Beneckea and in the mnethylotrophslMethylosinus trichosporium and Pseudomonas ex-

-torquens. If the same coefficient of 55 (for mono--haen cytochrome) is assumed in the present workfor comparative purposes, then it can be concludedthat 36% of the cytochrome CH and 72% of thecytochrome CL bind CO in CO-saturated solution at250C.

pH-dependence of the midpoint redox potentials ofcytochromes CH and CLThe midpoint potentials presented in Fig. 5 and

Table 3 show that cytochromes CH and CL are verysimilar in the response of their midpoint potentials tochanges in pH. The El values at pH 7.0 were

Vol. 192

(a) i a

550.5

400 450 500 550 600

(b)

to0I

550

V 420.5

400 450 500 550 600

Wavelength (nm)Fig. 4. (Reduced + CO)-minus-reduced difference spectra

ofcytochromes CH and CL(a) Cytochrome CH (3.5,M); (b) cytochrome CL(3.8,UM). Cytochromes were reduced with dithioniteand CO was bubbled through for 30min.

+294mV and +256 mV, and these values are typicalof the high-potential monohaem bacterial cyto-chromes (Lemberg & Barrett, 1973). The apparentEo values were +404mV and +345mV for cyto-chromes CH and CL respectively. These values are'apparent' because potentials were not measuredbelow pH4.0, and further dissociations below thispH would lead to much higher real E. values. Bothcytochromes have two ionizing groups in theoxidized and reduced forms, the pK values being

14

417

D. T. O'Keeffe and C. Anthony

Table 3. pH-dependence ofmidpoint redox potentials ofcytochromes CH and CLThese pK and Eo values are calculated from the data in Fig. S by using the equation given in the legend.

Cytochrome CHCytochrome CL

pKo03.53.6

PK025.55.6

PKrl4.54.4

PKr2 EO (mV)6.5 4046.4 345

due to the front (outer) propionate group in a morehydrophilic environment. Allocation of the pKvalues to propionate groups for these cytochromeswould be consistent with proposals for the cyto-chromes c2 of Rhodospirillaceae (Pettigrew et al.,1975, 1978) and for cytochrome c551 of Pseudo-monas aeruginosa (Moore et al., 1980).

46

t

b

2 3 4 5 6pH

U.

7 90 . .-

a

7 8 9 lo0

Fig. 5. pH-dependence ofthe midpoint redox potentials ofcytochromes CH and CL

Details of the methods are given in the Materials andmethods section. Oxidative and reductive titrationsgave identical results. 0, Midpoint potentials ofcytochrome CH; *, midpoint potentials of cyto-chrome CL. The dotted lines are theoretical curvescalculated from the equation:

t[H+/1H~2+Kr,i[HI++Kr, Kr2EO=Eo + 59 log H1+K[H+1+K01 K02]

where Kol and K02 are the dissociation constants forthe first and second ionizations in the oxidizedspecies and Kr1 and K,2 are the equivalentdissociation constants for the reduced species. ThepK (-log K) and Eo values assumed for calculationof the theoretical curves are given in Table 3.

about 3.5 and 5.5 in the oxidized forms and 4.5 and6.5 in the reduced forms. These similarities suggestthat the ionizable groups and the haem environ-ments are very similar in the two cytochromes.

If the dissociations arise from the haem, then thelikely candidates are the propionate side chains. Ifthe propionate groups are involved, then the higherof the pK values is likely to be due to the rear (inner)propionate group buried in the hydrophobic environ-ment of the haem cleft; the lower pK would then be

Discussion

The properties of the pure cytochromes c fromPseudomonas AM 1 are summarized in Table 2. Theprevious description of a single cytochrome c fromthis organism (Anthony, 1975) was an accidentalcomposite one, some properties being of cyto-chrome purified by one method and other propertiesbeing of cytochrome purified by an alternativemethod. Thus it was concluded that the 'single'cytochrome c had a high isoelectric point and a highmolecular weight. As shown in the present work, themajor cytochrome c has a high isoelectric point buta low molelcular weight (cytochrome CH), whereasthe minor component that binds to DEAE-cellulosehas a low isoelectric point and a high molecularweight (cytochrome CL). The larger cytochrome CL(mol.wt. 20 900) is unlikely to be a dimer ofcytochrome CH (mol.wt. 11000) because thesemolecular weights were determined by SDS/poly-acrylamide-gel electrophoresis, which dissociatesdimers (Swank & Burris, 1969). The minor compo-nents of cytochrome CL separating from it duringpreparative isoelectric focusing have very similarisoelectric points (all being between 3.9 and 4.3), andthese may be isocytochromes produced by de-amidation.

It is unlikely that cytochrome CH is a breakdownproduct of cytochrome CL because the proportion ofthe two cytochromes is the same whether they areseparated on DEAE-cellulose within 1 h of celldisruption or after 24 h.

If, as appears to be the case, the two cyto-chromes c of Pseudomonas AM1 are fully distinctcytochromes, then the lack of both of them in thecytochrome c-deficient mutant PCT76 (Anthony,1975) must be explained. That the mutation is not ina gene responsible for haem biosynthesis is shownby the normal concentrations of cytochromes b anda + a3 in the mutant (Anthony, 1975; Widdowson &Anthony, 1975). It is possible that the mutation is ina regulatory gene affecting the concentrations of two

1980

420

400

3801-

360 _

320k

300 -

2801-

260

418

-044

340-,..--...

.0

Two cytochromes c of Pseudomonas AM 1 - 419

different cytochromes c. One may note a similarobservation that both cytochromes c and cl were lostby a single mutation in Paracoccus denitrificans(Willison & John, 1979).

There are few full descriptions of pure cyto-chromes c from methylotrophs, and so extensivecomparisons are impossible. In Methylophilusmethylotrophus (an obligate methanol-utilizer) thereare at least three soluble cytochromes c, and thecharacterization of these is extensively described inthe following paper (Cross & Anthony, 1980). Thebest-characterized from an obligate methanotroph isthat of Methylosinus trichosporium (Tonge et al.,1975, 1977). This cytochrome c has been completelypurified; it probably has a high isoelectric point,because it does not bind to DEAE-cellulose duringthe purification process. It has a low molecularweight (13 000), one haem-group molecule, amidpoint redox potential of +310mV, an absorp-tion coefficient of 22.5 M-1 * cm-' and an absorptionmaximum at 551 nm.The physiological role of the two cytochromes c

of Pseudomonas AM 1 is not completely clear,although some information on this is discussedelsewhere (O'Keeffe & Anthony, 1980). It wasthought previously that, because mutant PCT76completely lacks cytochrome c but still grows on allsubstrates except methanol, ethanol and methyl-amine, cytochrome c may not function in theoxidation of NADH in Pseudomonas AM 1(Anthony, 1975; O'Keeffe & Anthony, 1978).However, it has since been suggested that underconditions of carbon-limitation cytochrome c maybe involved in the oxidation of all substrates (Keevil& Anthony, 1979). This conclusion leads to thepossibility that one of the soluble cytochromes cmay only be involved in the oxidation of methanol,whereas the other may be involved exclusively in theoxidation of other substrates in some growthconditions.

We thank the Science Research Council for financialsupport.

References

Ambler, R. P. (1963) Biochem. J. 89, 341-349Anthony, C. (1975) Biochem. J. 146, 289-298

Bamforth, C. W. & Quayle, J. R. (1978) Biochem. J. 169,677-686

Bartsch, R. G. & Kamen, M. D. (1960) J. Biol. Chem.235, 825-831

Cross, A. R. & Anthony, C. (1980) Biochem. J. 192,421-427

Duine, J. A., Frank, J. & de Ruiter, L. G. (1979) J. Gen.Microbiol. 115, 523-526

Dutton, P. L., Wilson, D. F. & Lee, C. P. (1970)Biochemistry 9, 5077-5082

Giirtler, L. & Horstman, H. H. (1970) Eur. J. Biochem.12, 48-57

Higgins, I. J. (1979) Int. Rev. Biochem. 21, 300-353Keevil, C. W. & Anthony, C. (1979) Biochem. J. 182,

71-79Lemberg, R. & Barrett, J. (1973) Cytochromes, pp.

265-306, Academic Press, London and New YorkMacLennan, D. G., Ousby, J. C., Vasey, R. B. & Cotton,

N. T. (197 1) J. Gen. Microbiol. 69, 395-404Moore, G. R., Pettigrew, G. W., Pitt, R. C. & Williams,

R. J. P. (1980) Biochim. Biophys. Acta 590, 261-271Netrusov, A. I. & Anthony, C. (1979) Biochem. J. 178,

353-360O'Keeffe, D. T. & Anthony, C. (1978) Biochem. J. 170,

561-567O'Keeffe, D. T. & Anthony, C. (1979) Proc. Soc. Gen.

Microbiol. 6, 71O'Keeffe, D. T. & Anthony, C. (1980) Biochem. J. 190,

481-484Pettigrew, G. W., Aviram, I. & Schejter, A. (1975)

Biochem. J. 149, 155-167Pettigrew, G. W., Bartsch, R. G., Meyer, T. E. & Kamen,

M. D. (1978) Biochim. Biophys. Acta 503, 509-523Rieske, J. S. (1967) Methods Enzymol. 10, 488-493Swank, R. T. & Burris, R. H. (1969) Biochim. Biophys.

Acta 180, 473-489Swank, R. T. & Munkres, K. D. (1971) Anal. Biochem.

39, 462-477Sweetser, P. B. (1967)Anal. Chem. 39, 979-982Tonge, G. M., Knowles, C. J., Harrison, D. E. F. &

Higgins, I. J. (1974) FEBS Lett. 44, 106-1 10Tonge, G. M., Harrison, D. E. F., Knowles, C. J. &

Higgins, I. J. (1975) FEBS Lett. 58, 293-299Tonge, G. M., Harrison, D. E. F. & Higgins, I. J. (1977)

Biochem. J. 161, 333-344van Verseveld, H. W. & Stouthamer, A. H. (1978) Arch.

Microbiol. 118, 13-20Weston, J. A. & Knowles, C. J. (1974) Biochim. Biophys.

Acta 305, 11-18Widdowson, D. & Anthony, C. (1975) Biochem. J. 152

349-356Willison, J. C. & John, P. (1979) J. Gen. Microbiol. 115,

443-450

Vol. 192