PRELIMINARY NOTES 667

Table I, which also demonstrates that hydrolapachol inhibition of succinate oxidation is released by 2,4-dinitrophenol. On the other hand, inhibition by 2-hydroxy-3(3, 7- dimethyloctyl)-I,4-naphthoquinone was not reversed in comparable experiments.

Thu~,;, reversal by uncoupling agents is not a unique property of inhibitors acting on the NADH-flavoprotein region of the respiratory chain (Amytal and gua- nidines). HQNO appears to affect both energy-conserving reactions and electron transport itself as it has been shown to inhibit electron transport in non-phos- phorylating preparations as well s. Failure to obtain reversal of inhibition by an uncoupling agent should not be cited as sufficient evidence that an inhibitor does not react with part of the energy-transfer apparatus. The irreversibility of antimycin inhibition should perhaps be discussed in the context of its extremely tight binding to its target compound when compared to, for example, Amytal.

The author is indebted to Professor E. C. SLATER and to Dr. J. M. TAGER for valuable discussions during this work. Samples of substituted naphthoquinones were obtained through the generosity of Professor L. FIESER and HQNO through that of Dr. J LIGHTBOWN. The author was the recipient of a post-doctoral fellowship from the Division of General Medical Sciences, U.S. Public Health Service.

Laboratory of Physiological Chemistry, University of Amsterdam,

Amsterdam (The Netherlands)

J. L. HOWLAND *

t W. C. HOLSMANN, M.D. Thesis, Klein Offset Drukker i j , A m s t e r d a m , 1958. 2 H. C. HEMKER, Biochim. Biophys. Acta, in t he press. 8 B. CHAliCE, G. HOLLUNGER AND B. HAGIHARA, Biochem. Biophys. Res. Commun., 8 (1962) 18o. 4 B. CHANCE AND G. HOLLUNGER, J. Biol. Chem., 238 (I963) 418. 5 B. CHA~ICE AND G. HOLLUNGBR, J. Biol. Chem., 238 (I963) 432. 6 F. HUIJING AND E. C. SLATER, J. Biochem. Tokyo, 49 (1961) 493- 7 R. W. F.STABROOK, Biochim. Biophys. Acta, 6o (1962) 236. s j . W. LIGHTBOWN AND F. L. JACKSON, Biockem. J., 63 (1956) I3O.

Received May I5th, 1963

* P re sen t address : D e p a r t m e n t of Biology, Bowdoin College, Brunswick , Maine (U.S.A.).

Biochim. Biophys. .4aa, 73 (1963) 665-667

PN 1005O Reactions of cytochromes a and a 3 in the succinate oxidase system

Althoug:h KEILI~ AND HARTREE 1 in 1939 demonstrated the peculiarities in the spectrum of cytochrome a that can best be attributed to a component a 3, the distinct- ness of cytochromes a and a3 has only been accepted by some workers z-4. Others have at tempted to explain the "spectral anomalies" in terms of a single haemo- protein 5-7, invoking an interaction with copperS, e or complexes with oxygen 7 to explain the data indicating the existence of cytochrome a 3.

The; latter concept is buttressed by the fact that direct spectra of the absorption band of reduced a 3 have not been published. The spectrum of cytochrome aa ~+ CO

Biochim. Biophys. Acta, 73 (1963) 667-67o

668 PRELIMINARY NOTES

is known only as a difference spectrum (a*+a3*+CO minus a2+a38+HCN) and from the photochemical action spectrum s. But KEILIN AND HARTREE x reported tha t "on shaking the preparat ion containing CO with air, the component a m a y undergo oxidation while a 3 remains almost completely reduced and combined with CO", concluding tha t the oxidation of the other cytochromes m a y proceed via the small amount of uncombined a 3.

In contrast to such spectroscopic observations a spectrophotometric analysis

A A ~ M l I I I l i I I

0.06

0.05 A

0.04

0 . 0 3

0.02 E , ,, /

/ %

O.Ol

0.00

-0.01 620 6()0 ' 5~K) ' .5;0 ' 540 Wavelength (m/J)

Fig. i. Absorption spectra of cytochrome as2+CO in a heart-muscle preparation. 3.3 ml heart- muscle preparation (7 mg ml) in o.x 5 NI phosphate (pH 7.4)- A, plus i.z mM succinate under CO; B, plus 1.2 mM succinate in presence ofo.8 mM HCN in air; C, A minus B; D, A plus 2 mM KsFe(CN),; E: plus 3/zM succinate under CO. 'Blank' cell in each case contained oxidized heart- muscle preparation. Ass 0 mu has been arbitrarily set at o.oo for Spectra A to E (to eliminate scattering errors). Hilger Uvispek spectrophotometer with glass prism and high-intensity lamp

was used, with the cuvette about 4 cm from the photocell and a slit width of o.2 ram.

in the presence of oxygen is difficult. Thus the addition of oxygen to a reduced cytochrome (a + a3) preparat ion in CO causes an increasingly rapid dissociation of CO and oxidation of "as", followed by the oxidation of "a ''s.

In the experiments summarised in Fig. I , a KEILIN-HARTREE heart-muscle preparat ion was contained in an optical cuvet te a t tached to a Thunberg tube, and varying amounts of potassium ferricyanide or succinate were added from the stopper, with carbon monoxide as the gas phase.

Spectrum A (excess of succinate added initially) is typical of the cytochrome system in the presence of CO. The band of the oxidase-CO complex is at 605 In# with a "shoulder" at about 59 ° m/z. The difference between this and the aerobic cyanide- t reated preparat ion (B) is given by Spectrum C, which corresponds to the ass+CO difference spectrum for the purified oxidase (B of Fig. 8 in ref. 4). The addition of 2.0 mM ferricyanide to such a preparat ion gives Spectrum D. Here cytochrome b is total ly and cytochrome c partially oxidized; the band at 605 In# has become a "shoulder" and a peak occurs at 59 ° m/z. Similarly in Spectrum E, where a very small amount of succinate has been added to the initially oxidized system, only a

Biochim. Biophys. Acta, 73 (I963) 668~569

PRELIMINARY NOTES 669

59o-m# band is present. The marked resemblance of Spectra E and D to the cal- culated spectrum of a32+C0 (C) is evident.

The results may be interpreted in terms of the 'a-a 3' concept as follows: (a) a32+C0 is much less sensitive to ferricyanide than are a 2+, b 2+ and c 2+. A similar conclusion has been reached by HORIE AND MORRISON 1° using the isolated cyto- chrome (a + a3) system. (b) reducing equivalents added to the respiratory chain accumulate first at the cytochrome a3 ~+ level in the presence of CO.

I t is impossible to explain the results of Fig. I in terms of the 'A-Cu' conceptS, 6 unless some new interpretation of the 6o5-m# band is proposed. I f this were due to reduced 'cytochrome A' (e.g. where cyanide is inhibiting the final oxidation), it could not be oxidized when something remained complexed to CO as in D and E (Fig. i).

GIBSON AND GREENWOOD 9 recently showed that only some 35-5o% of the a haem may be t i t rated with CO in oxidase preparations. The succinate oxidase ac- t ivi ty of a cytochrome c-deficient heart-muscle preparation, treated with cyanide to eliminate the irreversible cyanide-binding groups n and then dialysed, may be partially inhibited by the addition of amounts of cyanide stoichiometric with the respiratory components. Preliminary calculations suggest that the cyanide binding sites (a3a+?) also represent only a fraction of the available haem a ( 4 5o%), i.e. like the CO-binding sites, less than either the haem a or copper content of the oxi- dase.

A cytochrome oxidase preparation (method of OKUNUKI eta/. 1~) treated with cytochrome c and ascorbate in air, in presence and absence of the inhibitors azide and sulphide, showed over a period of several hours only a diminished A 445 mu:A 805 m u

ratio in the inhibited systems, similar to that found in the case of cyanide 4. No in- dication of NO ferrous derivatives, or "sulph-" compounds, such as are formed by catalase and myoglobin13,14, could be seen. The addition of azide and sulphide to the complete succinate oxidase system similarly gives rise to no unusual absorption bands. The existence of oxyferrous 7 or ferryP 5 forms of a 3 is thus doubtful.

I t is possible to conclude tentatively: (a) that electrons may be transferred to a33+ from other aa 2+ haems le, from a s+ haems 9 (including those distant from the as a+ concerned1), from cytochrome c (probably indirectly)17, and possibly from other redox groups (such as Cu)9,1s; (b) that cytochromes a 8 and a are different entities, pre-existing in the succinate oxidase chain before the reactions with CO or O3 (cf. lack of proportionality between a-bands and concentrations), probably associated with different proteins (cf. presence of 28o-m# peak and absence of 6o5-m# peak in the photochemical action spectrum).

I should like to thank Dr. M. MORRISON for allowing me to see his communica- tion 1° before publication. I must also acknowledge here my debt to the ideas and stimulus of the late Professor D. KEILIN, a debt that cannot be repaid.

Molteno Institute, University of Cambridge, Cambridge (Great Britain)

PETER NICHOLLS

1 D. KEILIN AnD E. F. HARTREE, Proc. Roy. Soc. London, Ser. B, x27 (1939) 167. 2 B. CHANCE, J. Biol. Chem., 2o2 (1953) 383 • a E. C. SLATER, Advan. Enzymol., 2o (1958) 147.

Biochim. Biophys. Acta, 73 (1963) 667-670

670 PRELIMINARY NOTES

4 T. YONETANI, J. Biol. Chem., 235 (1963) 845. 5 D. E. GRIFFITHS AND D. C. WHARTON, J. Biol. Chem., 236 (1961) 1857. e W. W. WAINIO, J. Biol. Chem., 212 (1955) 723 . ? K. OKUNUKI, B. HAGIHARA, I. SEKUZU AND T. HORIO, Proe. Intern. Symp. Enzyme Chem.

Tokyo Kyoto, 1957, (z958), p. 264. s T. YONETANI AND G. W. KIDDER, J. Biol. Chem., 238 (1963) 386. 0 Q. H. GIBSON AND C. GREENWOOD, Biochem. J., 86 (1963) 541.

z0 S. HORIE AND M. MORRISON, Federation Proc., 22 (1963) Abstr. 2543. lZ C. P. LEE AND T. E. KING, Biochim. Biophys. Aeta, 59 (1962) 716. 1~ K. OKUNUKI, I. SEKUZU, T. YONETANI AND S. TAKEMORI, J . Biochem. Tokyo, 45 (1958) 847. 13 p. NICHOLLS, Bioehim. Biophys. Acta, 58 (1962) 386. z4 p. NICHOLLS, Biochem. ]., 81 (1961) 374- 15 K. MINNAERT, Biochim. Biophys. Aeta, 35 (1959) 282. 16 B. CHANCE AND T. YONETANI, Federation Proe., 18 (1959) 2o2. 17 T. YONETANI, J. Biol. Chem., 235 (i96o), 3138. as K. MINNAERT, Federation Proc., 2o Abstr. (1961) p. 42.

Received April Ist, 1963 Biochim. Biophys. Acta, 73 (1963) 667-670

PN Ioo42 Peptides from a tryptic digest of baker's yeast cytochrome c

I t is of interest to compare the amino acid sequence of cytochrome c from baker's yeast with that from mammalian sources, since the latter enzyme is replaceable by the former in the electron-transport system of mammals. Complete amino acid sequences of equine I and human 2 heart cytochrome c have been reported but no systematic study has been carried out for the yeast enzyme.

In the present communication, the fractionation of the peptides derived from the tryptic digest of baker's yeast cytochrome c and the structures of the purified peptides are described. Chromatographically purified cytochrome c was digested with trypsin at pH 8.0 and 3 o° for 3.5 h. The digest was fractionated by an Amber- lite CG-5o column using volatile pyridine-acetic acid buffer as is shown in Fig. I. Several peaks were not homogeneous and they were purified by paper electrophoresis (pH 3.6, pyridine-acetic acid, 2000-3000 V, I-2 h) or paper chromatography (mainly n:butanol-pyridine-acetic acid-water, I5 : Io :3 : I2 ). Amino acid sequences of the purified peptides were elucidated by the usual terminal analysis methods. Large peptides were split by chymotrypsin, pepsin, hydrochloric acid or cyanogen bromide if methionine is contained. Deduced structures of these peptides are summarized in Table I.

Peptide IV must be the N-terminal peptide, since the N-terminal sequence, Thr-Glu-Phe, was already established 3. Peptides I - I and I-2 contain no basic C- terminal residue and seem to be the C-terminal peptides. Peptide I - I is a mixed disulphide of Lys-Ala-CyS-Glu and Ala-CyS-Glu and appears to be produced during the digestion and fractionation. Peptides I I I - i , V, VI-I , VI-2 and IX seemed to be derived from the same part of the peptide chain and could be combined to the following sequence : Lys-Glu-Lys-Asp-Arg-Asp(NH,)-Asp-Leu-I leu-Thr-Tyr-Leu- Lys. Amino acid compositions of Peptides 111-2 and VI-4 were the same and one of which might be a modified form of the other. Yields of Peptides XIV and XV

Bioehim. Biophys. Aeta, 73 (1963) 67o-673