Electron Transporting Components Participating in Nitrate and Oxygen Respirations from a Halotolerant Micrococcus

The Journal of Biochemistry, Vol. 50, No. 6, 1961

II. Properties of Cytochrome c551 and Brown Protein

By KATSUJI HORI

(From the Department of Chemistry, Faculty of Science, Nagoya Univercity, Nagoya)

(Received for publication, July 13, 1961)

In a previous paper of this series, the ing to the method of Davenport and Hill (2).

methods for purification of five chromopro Spectrophotometric observations were made by means

teins, that is, cytochrome c551, brown protein, of a Hitachi model EPU-2 Spectrophotometer using

cytochrome b4 (I) and b4 (II) and HdR* 1cm. cells. Protein was determined by the method of Lowry et al. (3). (cytoehrome 625, 553) were described (1). Characterization of cytochromes b4 (I) and b4 RESULTS (II) was also described there in detail. The Spectroscopic Properties of Cytochrome 0551 and purpose of this comunication is to describe the Brown Protein•\Cytochrome c551 and the the properties of cytochrome c551 and the brown brown protein were purified by the column protein. These chromoproteins were isolated chromatography on DEAE-cellulose, which from a halotolerant denitrifying Micrococcus.

Cytochrome c551 resembled mammalian cy

tochrome c in absorption spectrum and oxida

tion-reduction potential which was found to

be approximately +0.249 volt., but had a

lower isoelectric point than the latter. Che

mical nature and physiological function of

the brown protein is obscure at present.

MATERIALS AND METHODS

Bacterial Culture•\A halotolerant denitrifying Micro coccus, stain No. 203 was used. Cultivation of the

bacteria,** extraction and purifiication of chromo

proteins were performed, by methods described in the previous paper (1). Chemical and Physical Determinations•\Sedimentation

studies were performed with Spinco mode E ultra centrifuge. Electrophoretic mobilities for cytochrome

c551 and the brown protein were determined with a Hitachi Tiselius type electropholesis apparatus using

0.05M phosphate buffer, pH 7.0 at 4•Ž. The oxida-

tion-reduction protentials were measured at 25•Ž at

pH 7.0 using ferricyanide•\ferrocyanide system accord-

* Abbreviations :HeiR, hydroxylamine reductase ; DEAE-cellulose

, N,N'-diethyl am inoethyl-cellurose. ** The large scale cultivation of the bacteria was FIG. 1. Absorption spectra of cytochromt c551.•\ carried out by courtesy of Nagoya Factory of Fujisawa : Reduced form (with Na2S2O4), ...:O xidized form. Chemicals Co. Ltd. 482 K. HORI

was previously equilibrated with 0.1M am Sedimentation Constants of cytochrome 011 and

monium acetate of pH 6.0. The absorption the Brown Protein•\Measurements of sedimenta spectra of partially purified cytochrome c551 tion constant were performed at a single

and the brown protein are shown in Figs. 1 concentration of 0.36per cent for cytochrome and 2. In visible regions there were maxima c551 and of 0.45per cent for the brown protein at 551, 521 and 416mƒÊ in reduced form, and in 0.05M phosphate buffer, pH 7.0. The

521 and 411mƒÊin oxidized form of cyto sedimentation diagram showed a single sedi chrome c551, the ratios of the extinctions of ƒ¿ to mentating boundary for each chromoprotein ƒÀ bands and Soret to ƒ¿ bands of the cyto- and the sedimentation constants of cytochrome c551 and the brown protein were 1.3S and 1.6 S,

respectively.

Electrophoretic Mobilities of cytochrome c551

and the Brown Protein•\-Cytochrome c551 showed

a single schlieren peak, while the schlieren

pattern of the brown protein indicated the exis tence of two components which consisted of

a faster moving brown colored component

and a slower moving colorless impurity.

Electrophoretic mobility of 10.9•~l0-5cm.2

sec-1volt.-1 was obtained for cytochrome c551

Oxidation-reduction Potentials of cytochrome

FIG. 2. Absorption spectra of the brown pro. em and the Brown Protein•\Oxidation-reduction

tein. •\•\: Reduced form (Na2S2O4), ----- potentials for these chromoproteins were Oxidized form, ... : Oxidized form+KCN. measured at 25•Ž and at pH 7.0. The E0' of

cytochrome c551 was +0.249 volt. while that

TABLE I for the brown protein was +0.36 volt. These Spectroscopic and Physicochemical Properties of values were caluclated from the data shown cytochrome c551 and the Brown Protein in Figs. 3 and 4 by means of the following,

equation, where the value 0.43 is the mid

point potential of ferricyanide-ferrocyanide system (2), and Eo' is the mid-point potential

of cytochrome c551 or the brown protein systems.

chrome are shown in Table I. On the other hand, the brown protein exhibited an absorp FIG. 3. Determinations of the oxidation- tion spectrum with maximum at 415mƒÊ in reduction potential of cytochrome c551 (•›) and

reduced form and 408mƒÊ in oxidized form, mammalian cytochrome c (•¢).

and apparently no other band has detectable Study carried out at 25•Ž at pH 7.0 in the

in visible region. presence of the ferricyanide-ferrocyanide system. Electron Transporting Components . II 483

previous paper, cytochrome c551 and the brown protein were isolated from the Micro coccus grown in aerobic and in anaerobic conditions with nitrate. Under anaerobic condition, the absolute amount of cytochrome c551 was reduced (1), whereas that of the brown protein remained unchanged. The greater part of cytochrome c551 was found in subcellular particles from which it was ex tracted with 40per cent of acetone without difficulty. cytochrome c551was easily reduced by succinate, formate and DPNH and was reoxidized by oxygen in the presence of the extracts of this Micrococcus. The initial rate FIG. 4. Determination of the oxidation of enzymatic reduction of this cytochrome reduction potential of the brown protein . was comparable to those of cytochromes b4 Study carried out at 25•Ž at pH 7.0 in the (I) and (II) while the initial rate of reoxida presence of the ferricyanide-ferrocyanide system. tion of reduced cytochrome c551 by oxygen

E0'(cytochrome)=0.43-0.59l was faster than those of cytochromes b4 (I) and b4 (II). It was comfermed the that reduced brown protein was capable of functioning as og K4Fe(CN)6/K3Fe(CN)6•~ferrocytochrome/ferrocytochrome Heme Group of cytochrome c551•\cytochrome an electron donor in the cytochrome oxidase c551 exhibited a typical c-type pyridine hemo system in place of cytochromes.

chromogen spectrum, in reduced form there DISCUSSION were maxima at 550, 520 and 415mƒÊ, and futhermore, the prosthetic group could not Five chromoproteins have been isolated be split from the protein by the treatment from a halotolerant denitrifying Micrococcus with HCl-acetone. and have been considerably purified by Chemical Nature of the Brown Protein•\As chromatography on DEAE-cellulose column.

yet no detailed analysis has been made of cytochrome c551and the brown protein were, iron and other metals in brown protein, but however, less acidic than the other cytochro no evidence for the presence of heme group mes, therefore a simple repetition of chroma has been obtained in the protein so far. tography on DEAE-cellulose column was not No shift of the absorption band at 408 so useful as expected for the purification of mƒÊ of oxidized form occurred on addition of these chromoproteins. Precipitation with ammonium sulfate and electrophoresis with potassium cyanide but the absorbancy dimi nished moderately (Fig. 2). Neither oxidized starch zone were available for further puri nor reduced form of the brown protein did fication of cytochrome 011 but the brown combine with carbon monoxide. These results protein seemed to break down during these suggested that this chromoprotein was diffe procedures. cytochrome c551 and the brown rent from the pigment called 'mitochrome' protein used in their characterizations were which was found in liver mitochondria, a of the purity of 80per cent and 60per cent, hemeprotein derived from the preparations respectively. As was shown in Fig. 1. and of cytochrome (a+a3) and of cytochrome Table I, cytochrome c551 resembled closely b(4), although the spectrum of the brown mammalian cytochrome c in spectroscopic

protein was in a close resemblance to mito property and in the oxidation-reduction po chrome. tential but seemed to have a lower isoelectric Enzymatic Properties of cytochrome c551 and point than mammalian cytochrome c. A the Brown Protein•\As was mentioned in the number of different cytochormes of type-c 484 K. HORI

similar to cytochrome c551 in spectroscopic nitrate, nitrite and other dissimilatory nitrate and physicochemical properties have been reduction products get to nitrogen gas. The isolated from a nitrogen fixing bacteria, respiratory chain of this Micrococcus had been Azotobacter vinelandii (5, 6) and also from deni presented by Chance (14), in which cyto trifying bacteria, Micrococcus denitrificans (7, 8), chromes b4 (I) and b4 (II) and CO-binding Pseudomonas denitrificans (7, 8,12) and Pseudomo pigment were included as terminal respiratory nas aeruginosa (9). These nitrogen fixing and pigment. denitrifying bacteria have been shown to Physiological functions of HdR and the contain several different cytochromes in ad brown protein remained to be determined. dition to a cytochrome resembled cytochrome Recently, however HdR was confirmed to csst of the Micrococcus, and recently a number function as soluble factor in particulate of cytochromes of type c and even of type b nitrite reductase system (15). (Micrococcus denitrificans and Pseudomonas deniti SUMMARYcytochrome ficans) were isolated from these bacteria (5, 6, 8-11). c551 and a brown protein In these bacteria, interrelationship of a have been obtained from a denitryfying Micro number of different cytochromes in oxygen coccus together with other cytochromes, that and nitrate respiration systems and in nitro is, cytochromes b4(I), b4(II) and cytochrome gen fixation system which also has been 625, 553 (HdR).cytochrome supposed as a form of respiration (13) are c551 resembled closely mam not yet clear.cytochrome malian cytochrme c in spectroscopic and in c551 was easily reduced by some other properties but seemed to have a succinate, formate and DPNH and was re lower isoelectric poiet than mammalian cyto oxidized by oxygen in the presence of an chrome c. extract of this Micrococcus. As was described Chemical nature of the brown protein in the paragraph treated upon enzymatic is not yet clear, and no evidence for the pre properties of cytochrome c551and the brown sence of heme group has been obtained in protein, the initial rate of enzymatic reduc this chromoprotein. tion of cytochrome cut was comparable to those of cytochromes b4 (I) and b4 (II) while The authors wishes to express his gratitude to Prof. F. Egami and Dr. S. Taniguchi for their the initial rate of reoxidation of reduced cyto valuable and encouraging advices and to Mr. E. chrome c551 by oxygen was fatser than to Itagaki for his discussion. The author also express those for cytochromes b4 (I) and b4(11). his thanks to Nagoya Factory of Fujisawa Chemicals It suggests that cytochrome c551 may func Co. Ltd. for the cultivation of the bacteria. tion a role similar to that of mammalian cytochromec. Interralationship of cytochrome REFERENCES 560*, cytochromes b4 (I) and b4 (II), cytochrome (1) Hori, K., J. Biochem., 50, 440 (1961) c551 and the brown protein participating in (2) Darvenport, H. E., and Hill, R., Proc. Roy. Soc. nitrate and oxygen respirations is not yet London, B 139, 327 (1952) clear at present. As was shown in Fig. 7 of (3) Lowry, O.H., Rosebrough, N.J., Farr, A. L., the previous paper (1), however, it is probable and Randall, R.J., J. Biol. Chem., 193, 265 that cytochrome 560, cytochromes b4 (I), b4(II) (1951) and c551 function as intermediate electron (4) Elliott, W. B., Hulsman, W. C., and Slater, E. C., carriers in the oxygen respiration system Biochimet Biophys.Acta, 33, 509 (1959) while in nitrate respiration system cytochrome (5) Tissieres, A., and Burris, R. H., Biochem. et Bio 560 and b4 (I) participate in carrying electron phys. Acta, 20, 436 (1956) from succinate, formate and DPNH toward (6) Neumann, N. P., and Burris, R. H., J. Biol. Chem., 234, 3286 (1959) * Most recently it was isolated from the Micro (7) Kamen, M. D., and Vernon, L. P. Biochim. et cocccusby use of sodium deoxycholate and lipase (1). Biophys. Acta, 17, 10 (1955) Electron Transporting Components. II 485

(8) Vernon, L. P., J. Biol. Chem., 222, 1035, 1045 (12) Iwasaki, H., J. Biochem., 47, 174 (1960) (1959) (13) Parker, C.A., and Scutt, P. B., Biochem, et Biophys. (9) Horio, T., J. Biochem., 45, 195, 267 (1958) Acta, 38, 230 (1960) (10) Tissieres, A., Biochem. J., 64, 582 (1956) (14) Chance, B., Discussions of the Farady Society, No. (11) Egami, F., Itahashi, M., Sato, R., and Mori, T., 20, 205 (1955) J. Biochem., 40, 527 (1953) (15) Taniguchi, S. et al., manuscript in preparation.

NOTE ADDED IN PROOF

It has been recently confirmed that, among the respiratory pigments obtained in soluble forms, the reduced brown protein was the best electron donor to the nitrate reductase (NaR) also isolated from this

Micrococcus. The NaR was purified by chromatography on DEAE-cellulose column free of cytochromes and cytochrome reductase and oxidase. The initial rate of reoxidation of the reduced brown protein by nitrate

was greater than that by oxygen in the presence of the Micrococcus particulate fraction; +‡™ •›. D. (at 408mƒÊ).

min.-1mg. Nitrogen-1, 0.210(NO3-) and 0.56 (O2), respectively. These results strongly indicate that the brown

protein may function as a direct electron donor to the NaR.