- Email: [email protected]
The formation and properties of a cytochrome c-type haemoprotein (cytochrome b2-haemoprotein-550) from cytochrome b2
BIOCHIMICA ET BIOPHYSICAACTA
349
BBA 65168 T H E FORMATION AND P R O P E R T I E S OF A CYTOCHROME c-TYPE HAEMOP R O T E I N (CYTOCHROME b2-HAEMOPROTEIN-55o ) FROM CYTOCHROME b2
R. K. MO:RTON (Deceased) AND KATHRYN SHEPLEY Departmerd of Agricultural Chemistry, Waite Agricultural Research Institute, University of Adelaide, Adelaide (Australia) (Received November ioth, 1964)
SUMMARY I. The riboflavin phosphate prosthetic group of cytochlome b2, the lactate dehydrogenase (L-lactate:cytochrome c oxidoreductase, EC I.I.2.3) of baker's yeast is irreversibly dissociated, and lactate dehydrogenase activity lost, during titration of the enzyme with p-chloromercuribenzoate or p-chloromercuriphenyl sulphonate. This treatment causes no apparent change in the protohaem prosthetic group, as the absorption maxima of reduced cytochrome b2 remain at 556.5, 528 and 423.5 m# and the haem may be dissociated with acetone-HC1 mixtures. 2. By precipitating the p-chloromercuriphenyl sulphonate-treated enzyme with acetone at --15% a new haemoprotein, named cytochrome b2-haemoprotein-55o, is produced. It has absorption maxima (reduced form) at 550, 52I and 415 m# and other characteristics of cytochrome c. Its pyridine haemochrome shows absorption maxima at 550, 521 and 414 m#, and its prosthetic group is no longer dissociated by acetone--HCl, but cleavage with silver sulphate-acetic acid yields a compound resembling haematohaemin as obtained from cytochrome c with identical treatment. The E o' of cytochrome b2-haemoprotein-55o at pH 7 is about +0.28 V. (cf. +o.12 V for cytcchrome b~-haem) and the haemoprotein is not auto-oxidizable or reactive with carbon monoxide. It has activity comparable with cytochrome c in reductase and oxidase enzyme systems, and is reduced by ascorbic acid. 3. Electrophoretic and ultracentrifugal analyses of cytochrome b~-haemoprotein-55o show a single component with zero mobility near pH 5, and tool. wt. 74 000-82 ooo, (cf. zero mobility near pH 5 and mol. wt. 177 ooo for the native, dimeric cytochrome b~). 4- Cytochrome b,-haemoprotein may also be produced by mixing the native enzyme, oxidized or reduced, with p-chloromercuriphenyl sulphonate at 37-70°; or with 5 M urea then p-chloromercuriphenyl sulphonate at 2 °. 5. It is concluded that one or both vinyl side-chains of cytochrome b2-haem become joined by thio-ether linkage to the protein. This could happen when a disulAbbreviations: PCMB, p-chloromercuribenzoate; PCMS, p-chloromercuriphenyl sulphonate; DCJP, 2,6-dichlorophenol indophenoL Biochim. Biophys. Aeta, 96 (1965) 349-356
35 °
R . K . MORTON, K. SHEPLEY"
phide bridge, or bridges, close to the haem becomes unmasked by heat, acetone or urea treatment, then cleaved by p-chloromercuriphenyl sulphonate.
INTRODUCTION
Cytochrome b~, the lactate dehydrogenase (L-lactate:cytochrome c oxidoreductase, EC 1.1.2.3) of baker's yeast contains equimolecular proportions of both riboflavin phosphate and of protohaem 1-3. Previous papers have described a haemoprotein derivative (cytochrome b,-haemoprotein-557) formed by removal of the ravin prosthetic group with acid ammonium sulphate, a flavoprotein derivative (cytochrome b2-flavoprotein) formed by removal of the haem prosthetic group with acetone-KCN, and an apoprotein derivative formed by removal of the two prosthetic groups with acetone-HC14, 5. Cytochrome b~ and haemoprotein-557 are typical protohaemoproteins with a-bands at 557 m/z in the ferrocompound. However, by appropriate treatment of the crystalline enzyme with p-chloromercuriphenyl sulphonate or with p-chloromercuribenzoate, a haemoprotein derivative is formed with an aband at 550 m/z in the ferrocompound. This derivative protein is called cytochrome b2-haemoprotein-55o; it resembles heart-muscle cytochrome c in a number of properties 4. This paper describes the preparation and some of the properties of the haemoprotein-55o, which appears to have thio-ether bonds between the protein and the haem group. MATERIALS AND METHODS
Type I-ferrocytochrome b2. Twice crystallised enzyme was prepared by the procedure of APPLEBY AND MORTONe as modified by MORTON AND SHEPLEY 7. Cytochrome c. Heart-muscle cytochrome c was prepared from horse-hearts according to KEILIN AND HARTREE8, or was obtained from Sigma Chemical Co. Absorption spectra. Optica CF 4 manual and recording spectrophotometers were used. Electrophoresis and sedimentation. These were carried out with the instruments and with the procedures described by ARMSTRONG et al. 9. Pyridine haemochromogen. This was formed with pyridine and alkali as described by A P P L E B Y AND MORTON 2. Lactate dehydrogenase activity. This was assayed with ferricyanide, cytochrome c or 2,6-dichlorophenol indophenol (DCIP) as electron acceptors 2.~. Cytochrome c oxidase. (EC 1.9.3.1 ). This was prepared from horse-heart muscle as described by KEILIN AND HARTREE 10, except that the precipitate was collected at p H 6.5 by centrifuging at about io ooo × g for 3 ° min at 2 °. NADH2-cytochrome c oxidoreductase (EC I.6.2.z). This was prepared from horse-heart according to RINGLER et al. n. Iron. This was determined colorimetrically with o-phenanthroline (SANDELLTM) after digestion of the protein with H,O2 (DRABKINI~). Protein. This was estimated as described by LOWRY et al. 14, with crystalline cytochrome b, as a standard. Biochim. Biophys. Acta, 96 (1965) 349-356
CYTOCHI~:OME b2-HAEMOPROTEIN-550
351
Chemicals. p-Chloromercuriphenyl sulphonate (PCMS) and p-chloromercuribenzoate (PCMB) were obtained from Sigma Chemical Co. Ltd., U.S.A. The concentration of PCMS was estimated from its extinction in o.oi M-sodium pyrophosphate (pH 6.8) by using eM2~ mu = 0.6- lO3, and e~a~24ma = 20" 103. EXPERIMENTAL AND RESULTS
Effect of P C M S and P C M B on cytochrome b2 APPLEBY AND MORTON1 showed that I mM PCMB displaced the flavin group and inhibited lactate dehydrogenase activity of cytochrome b2, and ARMSTRONG et al. 15 showed that PCMS and PCMB prevented aggregate formation in solutions of cytochrome b2 exposed to air. Since the effects of these two compounds were similar, PCMS was used in all future work because of its greater solubility. During further studies of these phenomena, approx. 30 mg of type I-ferrocytochrome b2 in IO ml of 0.3 M sodium lactate, 0.05 M sodium pyrophosphate, o.I mM EDTA (pH 6.8) was treated with I mM PCMS (final concentrati6n)at 2 ° for 48 h and then dialysed against 0.05 M sodium lactate, o.I mM EDTA (pH 6.8) for 48 h at 2 °. Whereas untreated type I-cytochrome b2 crystallises 'within a few hours under these conditions, no crystals were formed from the treated enzyme. However, between 400 and 600 m#, the absorption spectrum resembled that of type I-cytochrome b2, with a,/3 and ~-bands at 557, 528 and 423 m/,. Sodium lactate was added to adjust the lactate concentration of the dialysed material to o.I M, the solution was cooled to o °, and acetone at --15 ° was added slowly as the solution was cooled to --5 °. A salmon-pink crystalline precipitate formed at about 45% (v/v) of acetone. The crystals consisted of small plates, less regular and much smaller than those of cytochrome b2 formed under similar conditions (see MORTON 4, MORTON AND SHEPLEYT).The precipitate was collected by centrifuging at --5 °, washed with 50% (v/v) acetone-o.I M sodium lactate (pH 6.8), dissolved in o.I M sodium lactate, 0.05 M te~trasodium pyrophosphate, o.i mM EDTA (pH 6.8) and dialysed against 0.05 M sodium lactate, o.I mM EDTA, I mM magnesium sulphate (pH 6.8) at 2 ° for 14 h. The acetone supernatant remaining after precipitation of the protein was yellow and had a typical flavin fluorescence. Propertie,~ of cytochrome b2-haemoprotein-55o The~ absorption spectrum of the salmon-pink solution of this protein had maxima at 550, 521 and 415 m/,, corresponding with those of horse-heart cytochrome c. Addition of sodium dithionite caused no increase in the extinctions. At pH 6.8 CO had no effect on the absorption spectrum. The absorption spectrum of the oxidised material was obtained after oxidation by stepwise addition of IO mM ferricyanide until the a-band of the ferrohaemoprotein disappeared, and then dialysis against 0.05 M sodium pyrophosphate (pH 6.8). Table I shows a comparison of the extinction coefficients of horse-heart cytochrome c and of the haemoprotein derived from cytochrome bv Because of the position of the a-absorption band, henceforth this derived protein is called cytochrome b2-haemoprotein-55o. The ferrihaemoprotein-55o contained o.o4 moles of flavin per mole of haem and initially it was slowly reduced in the preser;tce of o.I M sodium lactate at pH 6.8; the ferricyanide reductase activity was about 9 ° units/mg. However, after several hours at room temp. in the presence Biochim. Biophys. ,4aa, 96 (1965) 349.356
352
R . K . MORTON, K. SHEPLEY
TABLE I COMPARATIVE SPECTRAL PROPERTIES OF CYTOCHROME b2-HAEMOPROTEIN-550 , CYTOCHROME C A N D T Y P E I-CYTOCHROME b 2
HORSE-HEART
M e a s u r e m e n t s were m a d e w i t h an O p t i c a CF 4 g r a t i n g s p e c t r o p h o t o m e t e r . E x t i n c t i o n v a l u e s are g i v e n for a s o l u t i o n c o n t a i n i n g I mM h a e m a n d for a l i g h t p a t h of I cm.
Haemoprotein550
Cytochrome c
Cytochrome be
55 ° 28 521 18 415 145 316 68 257 58o*
55o.o 27.8 521 17 415 13o 316 34 280 34
556.5 37 528 18 423.5 232 33 ° 52 264 20o
53 ° -41o -355 -257 58o"
53 ° 11 408 lO8 360 29 286 25
535 15 413 16o 359 48 263 18o
Ferro-compounds a-band E E- b and E ~-band E ~-band E Ultraviolet band E
Ferri-compounds E 7-band E E Ultraviolet band E
* T h e h i g h e x t i n c t i o n is due to b o u n d m e r c u r i p h e n y l s u l p h o n a t e .
of air, this activity was less than 5 units/mg; the initial reductase activity is attributed to residual cytochrome b2. The ferrihaemoprotein was rapidly reduced by ascorbic acid and cytochrome c reductase 11. With similar concentrations of ferricytochrome c and of the ferrihaemoprotein-55o, the rates of enzymic reduction were essentially the same. By HILL'S16 l~rocedure, the E o' at p H 7 in o.I M sodium pyrophosphate-HC1 buffer was estimated to be in the range +0.24-o.29 V. The extinction of a solution of ferrocytochrome c remained substantially unchanged when an approx, equimolecular amount (haem basis) of ferrihaemoprotein550 was mixed with it at p H 7. I t is likely, therefore, that the E o' values of the two compounds are very similar but it is possible that they do not interact with one another. The ferrohaemoprotein-55 o was not autoxidisable at p H 5-8, but was rapidly oxidised b y heart-muscle cytochrome c-oxidase 1°.
The prosthetic group of haemoprotein-55 o The absorption spectrum of the pyridine haemochrome of haemoprotein-55 o shows extinction maxima at 55o, 521 and 414 m/z, as in the pyridine haemochrome of heart-muscle cytochrome c. A sample of haemoprotein-55 o was dialysed against water for several hours. The iron content was estimated to be 0.o720/0 . A portion of the dialysed material was extracted with methylethylketone-HC1. The extract contained a very small amount Biochim. Biophys. Acta, 96 (1965) 349-356
CYTOCHROME b2-HAEMOPROTEIN-550
353
of protohaemin, as indicated b y an extinction m a x i m u m at 384 m#, and b y formation of a pyridine haemochromogen with a m a x i m u m extinction at 418 m/z. However, the amount extracted accounted for only about 12 % of the prosthetic group estimated to be pre.,,ent. A similar result was obtained on treatment with acetone-HC1. A portion of the dialysed haemoprotein-55o was therefore treated with acetoneHC1 at o ° and the precipitate was washed in acetone and suspended in about I ml of water. The suspension was treated with 0. 4 nil of acetic acid and 2 mg of silver sulphate and shaken at 4 °0 for I h. A sample of horse-heart ferroeytochrom3 c was treated similarly. Both preparations had maxima at 615 m#. Chromatography of the extracts cf both haemoprotein-55o and of cytochrome c showed similar components; the RF corresponded with that of haematohaemin. With haemoprotein-55o prepared from type II-ferrocytochrome b2 and PCMS at room te.mperature (see below) there was no protohaemin extractable with acetoneHCI, and the compound reacted similarly with silver sulphate in acetic acid to that described above.
Physico-c,~emical properties of haemoprotein-55 o Only one component was detected by ultracentrifuging, but a slight a s y m m e t r y of the boundary indicates the presence of a small amount of a component of higher molecular weight than the main component. The sedimentation coefficient (s°20.w) declined ~Lnearly with increasing protein concentration : s°,0,w is 3.6" lO -13 sec. The mol. wt. of 74 ooo was calculated from LANSINGANn KRAEMER'S17 equation applied to the results obtained in the equilibrium ultracentrifuge with both the original preparation of haemoprotein-55o from type I-ferrocytochrome b, and the preparation obtained from type II-ferrocytochrome b2 (see below). By moving-boundary electrophoresis in lactate-pyrophosphate buffer (I, about 0.6) and in lactate-KCl-imidazole buffer (I, about 0.2) only a single protein component was detected. At p H 6.2, 6.8 and 7.8, both the ascending and descending boundaries were symmetrical in both buffers for about 8 h. In one experiment in lactate pyrophosphate buffer at p H 6.8, after I I h some a s y m m e t r y of the boundary was apparent. From a plot of mobility versus pH, the protein was estimated to have zero mobility at p H 5.
The reaction of P C M S with cytochrome b2 Solutions of cytochrome b2 in o.i M sodium pyrophosphate, o.I mM E D T A (pH 6.8) were treated at 37 ° with either I or IO moles of PCMS/mole of haem; ferroand ferri-forms of both type I- and type II-cytochrome b~ were used, the former in the presence of 0.05 M sodium lactate. The changes in absorption spectra were followed with a recording spectrophotometer. Witlh type I- and type II-ferrocytochrome b2, the formation of cytochrome b 2ferrohaemoprotein-55o was indicated b y the progressive appearance of the typical spectrum of this compound. The rate was more rapid with the greater concentration of PCMB, but the same final extinction values at 55 ° m/z were obtained with both concentra~Lions. The final solutions were orange in colour and had a very strong r a v i n fluorescence; liberation of oxidized FMN was also indicated by the increase in absorption between 450 and 500 m#. Wit]a type I- and type II-ferricytochrome b2, the progress of the reaction was Biochim. Biophys. Acta, 96 (1965) 349-356
354
R . K . MORTON, K. SHEPLEY
indicated by the shift of the v-band from 416 m# (cytochrome b,) to 41o m# (haemoprotein-55o) and a decrease in the extinction. When no further change was apparent, the haemoprotein was reduced with sodium dithionite ; the spectrum of ferrohaemoprotein-55o was obtained. During the reaction of PCMS with ferricytochrome b~, flavin fluorescence appeared, as with the ferrocytochrome. With a solution of type I-ferrocytochrome b~ containing IO moles of PCMS/ mole of haem, the reaction at o ° was followed by observing the a- and r-bands of the haemoprotein with a low dispersion microspectroscope. After 3 days at o °, the aband had not changed from 557 mju, although the solution had a strong ravin fluorescence. On addition of acetone at o ° to this solution, the a-band rapidly migrated to 55 ° m/z, indicating formation of haemoprotein-55o. With only 0.5 mole of PCMS/ mole of haem at 37 °, spectroscopic observations indicated incomplete conversion of type I-ferrocytochrome b, to haemoprotein-55 o, since a-bands were clearly identifiable at 557 and 550 m# after several hours. A sample of type I-ferrocytochrome b~ in 0.3 M sodium lactate, 0.05 M sodium pyrophosphate, o.I mM EDTA (pH 6.8) was brought to 5 M urea and set aside for 48 h at 2 ° anaerobically. Then PCMS (19 mM final concentration) was added at 2 °. Spectroscopic observation indicated a progressive reaction to form haemoprotein-55o.
DISCUSSION
Cytochrome b, has protohaem as a prosthetic group but it differs considerably from other b-type cytochromes, especially in the magnitude of the coefficients of the principal extinction bands, (see MORTON18, Table V). This is not attributable to the presence of the FMN component, since the flavin-free derivative, cytochrome b~haemoprotein-557, has bands at almost identical wavelengths with similar elevated extinction coefficients4,5. The haemoprotein derivative described here was reported by MORTON4; it has a striking resemblance to heart-muscle cytochrome c in spectral and redox properties, and in reactivity with some enzymes. The prosthetic group is clearly not protohaem. The small amount of protohaem found in the first preparation may be attributed to some residual cytochrome b 2, the presence of which also accounts for the trace of ravin, the slight lactate dehydrogenase activity, and the slight asymmetry of the sedimenting boundary in the analytical ultracentrifuge. The failure to split from the protein with acetone-HCl, the cleavage with silver salt, and the identity of the product with that obtained from cytochrome c provide evidence that the prosthetic group of cytochrome b2-haemoprotein-55o is very similar to ferroporphyrin c and has at least one, and very probably two. thio-ether bonds to the protein. This conclusion is supported by the high reactivity of haemoprotein-55o with heart-muscle cytochrome c oxidase and with cytochrome c reductase, and by the marked change of E o' from about +o.12 V in cytochrome b, to about +0.28 V. In the absence of contrary evidence, it is tentatively concluded that the protohaem group of cytochrome b~ was converted to a ferroporphyrin c group, with thio-ether bonds to the protein. Extensive work has been directed toward the formation of thio-ether bonds from the vinyl side-chains of protoporphyrin and protohaem (see LEMBERG AND Biochim. Biophys. Acta, 96 (1965) 349-356
CYTOCHROMF b~-HAEMOPROTEIN-550
355
LEGGEI~). ZI~ILE2°, also ZEILE AND PIUTTI ~1, obtained compounds with an a-band at 55 o m# as a result of the vinyl side-chains of protohaem reacting with various amino acids, but thio-ether bonds were not formed. DEUL 2~, has described a reaction of 2,3 dimercaptopropanol with the vinyl group of protohaemoproteins in the presence of acetone. However, there appears to have been no previous account of a reaction forming a haemoprotein with thio-ether bonds to the polypeptide chain of the protein. Haemoprotein-55o has similar electrophoretic properties to cytochrome b2; it is apparently electrophoretically homogeneous and has zero mobility at about p H 5.o under the experimental conditions used. Horse-heart and yeast cytochromes c have iso-electric points of lO.65 (ref. 23) and 9.85 (ref. 24), respectively. Cytochrome b2 has I mole protohaem/82 ooo × g of protein2, 9. Since the extinction for 82 ooo × g of haemoprotein-55o is similar to that of 12 ooo x g of horseheart cytochrome c, it may be concluded that haemoprotein-55 o has I mole of haematin-c/82 ooo × g, and this is confirmed by the iron analysis. Whereas ferrocytochrom~: b2 has an S°2o,w value of 9.12 and a molecular weight from equilibrium sedimentatiLon of 177 300 (ref. 9), haemoprotein-55o has an S°2o,w value of 3.6o and a molecular weight from equilibrium sedimentation of 74 ooo. It is apparent, therefore, that the conversion of cytochrome b2 to haemoprotein-55o involves a reaction in which there is cleavage of the molecule. This reaction occurs in the presence of PCMS and of PCMB at 37 °, the rate increasing vcith temperature, and is almost instantaneous at 7 °0 or higher, but the protein is i hen rapidly denatured. It proceeds at a negligible rate at 0-2 ° except in the presence of acetone. The requirement of either an elevated temperature, or the presence ot! acetone at low temperature suggests that some unfolding of the protein, possibly involving rupture of hydrogen bonds, must precede the reaction with PCMS. This is supported b y the observation that haemoprotein-55 o is formed rapidly at o ° when PCMS is added to ferrocytochrome b~ in 5 M urea (p. 6). Although the specificity of the reaction has not been investigated, it is likely that other mercurials, such as methyl mercuric iodide, PCMB and possibly mercuric chloride react similarly to PCMS. Haemoprotein-55o was not formed by heating the enzyme in the presence of I M KCN, or of o.oi M 2,3-dimercaptopropanol. The reaction occurs with both ferri- and ferro-cytochrome b~. The shift of the maximum extinction in the ultraviolet region to about 257 m/~, and the very large increase in the extinction shows that the PCMS has combined with the protein and is still attached in haemoprotein-55o. Previously, ARMSTRONG et al. 25, have shown that there are four thiol groups/ haem of cytochrome b2 which react rapidly with PCMS. With 1-2 moles of PCMS/ mole of haem, the formation of oxygen-induced aggregates of the enzyme is prevented, ravin is displaced, and the dehydrogenase activity is substantially inhibited. This has been interpreted as indicating the presence of 1-2 thiol groups at the active centre of 1:he enzyme 2"~.Arsenite (io -2 M), Cu 2+ (lO-3 M), N-ethylmaleimide (IO-s M) all inhibit the enzyme substantially, supporting evidence for a thiol or thiol groups at the active centre, and this appears to be a thiol involved in binding of the ravin group. These observations are probably related to the formation of haemoprotein-55o, but are inadequate to explain it. There are, however, 18 half-cystine groups/mole of Biochim. Biophys. Acta, 96 (1965) 349-356
356
R . K . MORTON, K. SHEPLEY
haem in cytochrome b2 (ref. 26) and hence 14 half-cystine groups which apparently do not react with PCMS in the intact enzyme. Hence there are available a large number of potentially-reactive groups. Since the formation of haemoprotein-55o is accompanied by cleavage of the molecule of cytochrome b~, one possibility is that a disulphide bridge, close to the haem group, is cleaved and the thiols so formed attack the vinyl groups. CUNNINGHAM et al. ~v have reported a reaction of PCMB with disulphide bonds at elevated temperatures. Whatever the mechanism, it seems that the thiol (or disulphide) groups must initially be adjacent to the haem group of the enzyme. Further work will be directed toward elucidation of the mechanism of the reaction. Since cytochrome b~-haemoprotein-55o has covalent bonds between the polypeptide chain and the haem goup, this derivative should be suitable for studying the sequence of amino acids near the haem group. It is very unlikely that this sequence is modified from that present in cytochrome b, itself.
ACKNOWLEDGEMENTS
The assistance of Mr. R. CONNOLLYand the generous provision of substantial amounts of yeast by Messrs. Effront Yeast Co. Ltd., Melbourne, is gratefully acknowledged as is the grant-in-aid from the Nuffield Foundation. REFERENCES I C, A. APPLEBY AND R. K. MORTON, Nature, 173 (1954) 749. 2 C. A. APPLEBY AND R. K. MORTON, Biochem. J., 73 (1959) 539. 3 R. K. MORTON, J. McD. ARMSTRONG AND C. A. APPLEBY, Haematin Enzymes Syrup., Canberra, x959, P e r g a m o n Press, Oxford, 1961, p. 5Ol. 4 R. K. MORTON, Nature, 192 (1961) 727. 5 R. K. MORTON AND K. SHEPLEY, Biochem. Z., 338 (1963) 122. 6 C. A. APPLEBY AND R. K. MORTON, Biochem. J., 71 (1959) 492. 7 R. K. MORTON AND K. SItEPLEY, Biochem. J., 89 (1963) 257. 8 D. KEILIN AND E. F. HARTREE, Biochem. J., 39 (1945) 289. 9 J- McD. ARMSTRONG, J. H. COATES AND R. K. MORTON, Biochem. J. ,86 (1963) 136. IO D. KEILIN AND E. F. I-IARTREE, Biochem. J., 41 (1947) 5 °0. I I R . L . RINGLER, S. MINAKAMI AND T. P. SINGER, Biochem. Biophys. Res. Commun., 3 (196o) 417 . 12 E. ]3. SANDELL, Colorimetric Determination of Traces of Metals, Interscience, New York, 3rd edition, 1959, p. 54113 D. L. DRABKIN, J. Biol. Chem., 14o (1941) 387 . 14 O. H. LOWRY, N. J. ROSEBROUGH, A. L. FARR AND R . J . RANDALL, J. Biol, Chem., 193 (1951 ) 265. 15 J. McD. ARMSTRONg, J. H. COATES AND R. K. MORTON, Nature, 186 (196o) lO33. 16 R. HILL, in K. PAECH AND M. V. TRACEY, Modern Methods of Plant Analysis, Vol. I, SpringerVerlag, Berlin, 1956, p. 39317 W. D. LANSING AND E. O. KRAEMER, J. Am. Chem. Soc., 57 (1935) t369. 18 R. K. MORTON, Rev. Pure Appl. Chem., 8 (1958) 161. 19 R. LEMBERG AND J. W. LEGGE, Hematin Compounds and Bile Pigments, Interscience, N e w York, 1949, p. 2o4. 20 K. ZEILE, Z. Physiol. Chem., 207 (1932) 35. 21 K. ZEILE AND P. PIUTTI, Z. Physiol. Chem., 218 (1933) 52. 22 D. H. DEUL, Biochim. Biophys. Acta, 48 (1961) 242. 23 H. THEORELL AND A. ~kKESSON, J. Am. Chem. Sot., 63 (1941) 18o4. 24 J. McD. ARMSTRONG, J. H. COATES AND R. I~. MORTON, Haematin Enzymes Syrup., Canberra, 5959, P e r g a m o n Press, Oxford, 1961, p. 385 . 25 J. McD. ARMSTRONG, J. H. COATES AND R. K. MORTON, Biochem. J., 88 (1963) 266. 26 C. A. APPLEBY, R. K. MORTON AND D. H. SIMMONDS, Biochem. J., 75 (196o) 72. 27 L. W. CUNNINGHAM, B. J. NUENKE AND W. D. STRAYHORN, J. Biol. Chem., 228 (1957) 835.
Biochim. Biophys..4eta, 96 (1965) 349-356
349
BBA 65168 T H E FORMATION AND P R O P E R T I E S OF A CYTOCHROME c-TYPE HAEMOP R O T E I N (CYTOCHROME b2-HAEMOPROTEIN-55o ) FROM CYTOCHROME b2
R. K. MO:RTON (Deceased) AND KATHRYN SHEPLEY Departmerd of Agricultural Chemistry, Waite Agricultural Research Institute, University of Adelaide, Adelaide (Australia) (Received November ioth, 1964)
SUMMARY I. The riboflavin phosphate prosthetic group of cytochlome b2, the lactate dehydrogenase (L-lactate:cytochrome c oxidoreductase, EC I.I.2.3) of baker's yeast is irreversibly dissociated, and lactate dehydrogenase activity lost, during titration of the enzyme with p-chloromercuribenzoate or p-chloromercuriphenyl sulphonate. This treatment causes no apparent change in the protohaem prosthetic group, as the absorption maxima of reduced cytochrome b2 remain at 556.5, 528 and 423.5 m# and the haem may be dissociated with acetone-HC1 mixtures. 2. By precipitating the p-chloromercuriphenyl sulphonate-treated enzyme with acetone at --15% a new haemoprotein, named cytochrome b2-haemoprotein-55o, is produced. It has absorption maxima (reduced form) at 550, 52I and 415 m# and other characteristics of cytochrome c. Its pyridine haemochrome shows absorption maxima at 550, 521 and 414 m#, and its prosthetic group is no longer dissociated by acetone--HCl, but cleavage with silver sulphate-acetic acid yields a compound resembling haematohaemin as obtained from cytochrome c with identical treatment. The E o' of cytochrome b2-haemoprotein-55o at pH 7 is about +0.28 V. (cf. +o.12 V for cytcchrome b~-haem) and the haemoprotein is not auto-oxidizable or reactive with carbon monoxide. It has activity comparable with cytochrome c in reductase and oxidase enzyme systems, and is reduced by ascorbic acid. 3. Electrophoretic and ultracentrifugal analyses of cytochrome b~-haemoprotein-55o show a single component with zero mobility near pH 5, and tool. wt. 74 000-82 ooo, (cf. zero mobility near pH 5 and mol. wt. 177 ooo for the native, dimeric cytochrome b~). 4- Cytochrome b,-haemoprotein may also be produced by mixing the native enzyme, oxidized or reduced, with p-chloromercuriphenyl sulphonate at 37-70°; or with 5 M urea then p-chloromercuriphenyl sulphonate at 2 °. 5. It is concluded that one or both vinyl side-chains of cytochrome b2-haem become joined by thio-ether linkage to the protein. This could happen when a disulAbbreviations: PCMB, p-chloromercuribenzoate; PCMS, p-chloromercuriphenyl sulphonate; DCJP, 2,6-dichlorophenol indophenoL Biochim. Biophys. Aeta, 96 (1965) 349-356
35 °
R . K . MORTON, K. SHEPLEY"
phide bridge, or bridges, close to the haem becomes unmasked by heat, acetone or urea treatment, then cleaved by p-chloromercuriphenyl sulphonate.
INTRODUCTION
Cytochrome b~, the lactate dehydrogenase (L-lactate:cytochrome c oxidoreductase, EC 1.1.2.3) of baker's yeast contains equimolecular proportions of both riboflavin phosphate and of protohaem 1-3. Previous papers have described a haemoprotein derivative (cytochrome b,-haemoprotein-557) formed by removal of the ravin prosthetic group with acid ammonium sulphate, a flavoprotein derivative (cytochrome b2-flavoprotein) formed by removal of the haem prosthetic group with acetone-KCN, and an apoprotein derivative formed by removal of the two prosthetic groups with acetone-HC14, 5. Cytochrome b~ and haemoprotein-557 are typical protohaemoproteins with a-bands at 557 m/z in the ferrocompound. However, by appropriate treatment of the crystalline enzyme with p-chloromercuriphenyl sulphonate or with p-chloromercuribenzoate, a haemoprotein derivative is formed with an aband at 550 m/z in the ferrocompound. This derivative protein is called cytochrome b2-haemoprotein-55o; it resembles heart-muscle cytochrome c in a number of properties 4. This paper describes the preparation and some of the properties of the haemoprotein-55o, which appears to have thio-ether bonds between the protein and the haem group. MATERIALS AND METHODS
Type I-ferrocytochrome b2. Twice crystallised enzyme was prepared by the procedure of APPLEBY AND MORTONe as modified by MORTON AND SHEPLEY 7. Cytochrome c. Heart-muscle cytochrome c was prepared from horse-hearts according to KEILIN AND HARTREE8, or was obtained from Sigma Chemical Co. Absorption spectra. Optica CF 4 manual and recording spectrophotometers were used. Electrophoresis and sedimentation. These were carried out with the instruments and with the procedures described by ARMSTRONG et al. 9. Pyridine haemochromogen. This was formed with pyridine and alkali as described by A P P L E B Y AND MORTON 2. Lactate dehydrogenase activity. This was assayed with ferricyanide, cytochrome c or 2,6-dichlorophenol indophenol (DCIP) as electron acceptors 2.~. Cytochrome c oxidase. (EC 1.9.3.1 ). This was prepared from horse-heart muscle as described by KEILIN AND HARTREE 10, except that the precipitate was collected at p H 6.5 by centrifuging at about io ooo × g for 3 ° min at 2 °. NADH2-cytochrome c oxidoreductase (EC I.6.2.z). This was prepared from horse-heart according to RINGLER et al. n. Iron. This was determined colorimetrically with o-phenanthroline (SANDELLTM) after digestion of the protein with H,O2 (DRABKINI~). Protein. This was estimated as described by LOWRY et al. 14, with crystalline cytochrome b, as a standard. Biochim. Biophys. Acta, 96 (1965) 349-356
CYTOCHI~:OME b2-HAEMOPROTEIN-550
351
Chemicals. p-Chloromercuriphenyl sulphonate (PCMS) and p-chloromercuribenzoate (PCMB) were obtained from Sigma Chemical Co. Ltd., U.S.A. The concentration of PCMS was estimated from its extinction in o.oi M-sodium pyrophosphate (pH 6.8) by using eM2~ mu = 0.6- lO3, and e~a~24ma = 20" 103. EXPERIMENTAL AND RESULTS
Effect of P C M S and P C M B on cytochrome b2 APPLEBY AND MORTON1 showed that I mM PCMB displaced the flavin group and inhibited lactate dehydrogenase activity of cytochrome b2, and ARMSTRONG et al. 15 showed that PCMS and PCMB prevented aggregate formation in solutions of cytochrome b2 exposed to air. Since the effects of these two compounds were similar, PCMS was used in all future work because of its greater solubility. During further studies of these phenomena, approx. 30 mg of type I-ferrocytochrome b2 in IO ml of 0.3 M sodium lactate, 0.05 M sodium pyrophosphate, o.I mM EDTA (pH 6.8) was treated with I mM PCMS (final concentrati6n)at 2 ° for 48 h and then dialysed against 0.05 M sodium lactate, o.I mM EDTA (pH 6.8) for 48 h at 2 °. Whereas untreated type I-cytochrome b2 crystallises 'within a few hours under these conditions, no crystals were formed from the treated enzyme. However, between 400 and 600 m#, the absorption spectrum resembled that of type I-cytochrome b2, with a,/3 and ~-bands at 557, 528 and 423 m/,. Sodium lactate was added to adjust the lactate concentration of the dialysed material to o.I M, the solution was cooled to o °, and acetone at --15 ° was added slowly as the solution was cooled to --5 °. A salmon-pink crystalline precipitate formed at about 45% (v/v) of acetone. The crystals consisted of small plates, less regular and much smaller than those of cytochrome b2 formed under similar conditions (see MORTON 4, MORTON AND SHEPLEYT).The precipitate was collected by centrifuging at --5 °, washed with 50% (v/v) acetone-o.I M sodium lactate (pH 6.8), dissolved in o.I M sodium lactate, 0.05 M te~trasodium pyrophosphate, o.i mM EDTA (pH 6.8) and dialysed against 0.05 M sodium lactate, o.I mM EDTA, I mM magnesium sulphate (pH 6.8) at 2 ° for 14 h. The acetone supernatant remaining after precipitation of the protein was yellow and had a typical flavin fluorescence. Propertie,~ of cytochrome b2-haemoprotein-55o The~ absorption spectrum of the salmon-pink solution of this protein had maxima at 550, 521 and 415 m/,, corresponding with those of horse-heart cytochrome c. Addition of sodium dithionite caused no increase in the extinctions. At pH 6.8 CO had no effect on the absorption spectrum. The absorption spectrum of the oxidised material was obtained after oxidation by stepwise addition of IO mM ferricyanide until the a-band of the ferrohaemoprotein disappeared, and then dialysis against 0.05 M sodium pyrophosphate (pH 6.8). Table I shows a comparison of the extinction coefficients of horse-heart cytochrome c and of the haemoprotein derived from cytochrome bv Because of the position of the a-absorption band, henceforth this derived protein is called cytochrome b2-haemoprotein-55o. The ferrihaemoprotein-55o contained o.o4 moles of flavin per mole of haem and initially it was slowly reduced in the preser;tce of o.I M sodium lactate at pH 6.8; the ferricyanide reductase activity was about 9 ° units/mg. However, after several hours at room temp. in the presence Biochim. Biophys. ,4aa, 96 (1965) 349.356
352
R . K . MORTON, K. SHEPLEY
TABLE I COMPARATIVE SPECTRAL PROPERTIES OF CYTOCHROME b2-HAEMOPROTEIN-550 , CYTOCHROME C A N D T Y P E I-CYTOCHROME b 2
HORSE-HEART
M e a s u r e m e n t s were m a d e w i t h an O p t i c a CF 4 g r a t i n g s p e c t r o p h o t o m e t e r . E x t i n c t i o n v a l u e s are g i v e n for a s o l u t i o n c o n t a i n i n g I mM h a e m a n d for a l i g h t p a t h of I cm.
Haemoprotein550
Cytochrome c
Cytochrome be
55 ° 28 521 18 415 145 316 68 257 58o*
55o.o 27.8 521 17 415 13o 316 34 280 34
556.5 37 528 18 423.5 232 33 ° 52 264 20o
53 ° -41o -355 -257 58o"
53 ° 11 408 lO8 360 29 286 25
535 15 413 16o 359 48 263 18o
Ferro-compounds a-band E E- b and E ~-band E ~-band E Ultraviolet band E
Ferri-compounds E 7-band E E Ultraviolet band E
* T h e h i g h e x t i n c t i o n is due to b o u n d m e r c u r i p h e n y l s u l p h o n a t e .
of air, this activity was less than 5 units/mg; the initial reductase activity is attributed to residual cytochrome b2. The ferrihaemoprotein was rapidly reduced by ascorbic acid and cytochrome c reductase 11. With similar concentrations of ferricytochrome c and of the ferrihaemoprotein-55o, the rates of enzymic reduction were essentially the same. By HILL'S16 l~rocedure, the E o' at p H 7 in o.I M sodium pyrophosphate-HC1 buffer was estimated to be in the range +0.24-o.29 V. The extinction of a solution of ferrocytochrome c remained substantially unchanged when an approx, equimolecular amount (haem basis) of ferrihaemoprotein550 was mixed with it at p H 7. I t is likely, therefore, that the E o' values of the two compounds are very similar but it is possible that they do not interact with one another. The ferrohaemoprotein-55 o was not autoxidisable at p H 5-8, but was rapidly oxidised b y heart-muscle cytochrome c-oxidase 1°.
The prosthetic group of haemoprotein-55 o The absorption spectrum of the pyridine haemochrome of haemoprotein-55 o shows extinction maxima at 55o, 521 and 414 m/z, as in the pyridine haemochrome of heart-muscle cytochrome c. A sample of haemoprotein-55 o was dialysed against water for several hours. The iron content was estimated to be 0.o720/0 . A portion of the dialysed material was extracted with methylethylketone-HC1. The extract contained a very small amount Biochim. Biophys. Acta, 96 (1965) 349-356
CYTOCHROME b2-HAEMOPROTEIN-550
353
of protohaemin, as indicated b y an extinction m a x i m u m at 384 m#, and b y formation of a pyridine haemochromogen with a m a x i m u m extinction at 418 m/z. However, the amount extracted accounted for only about 12 % of the prosthetic group estimated to be pre.,,ent. A similar result was obtained on treatment with acetone-HC1. A portion of the dialysed haemoprotein-55o was therefore treated with acetoneHC1 at o ° and the precipitate was washed in acetone and suspended in about I ml of water. The suspension was treated with 0. 4 nil of acetic acid and 2 mg of silver sulphate and shaken at 4 °0 for I h. A sample of horse-heart ferroeytochrom3 c was treated similarly. Both preparations had maxima at 615 m#. Chromatography of the extracts cf both haemoprotein-55o and of cytochrome c showed similar components; the RF corresponded with that of haematohaemin. With haemoprotein-55o prepared from type II-ferrocytochrome b2 and PCMS at room te.mperature (see below) there was no protohaemin extractable with acetoneHCI, and the compound reacted similarly with silver sulphate in acetic acid to that described above.
Physico-c,~emical properties of haemoprotein-55 o Only one component was detected by ultracentrifuging, but a slight a s y m m e t r y of the boundary indicates the presence of a small amount of a component of higher molecular weight than the main component. The sedimentation coefficient (s°20.w) declined ~Lnearly with increasing protein concentration : s°,0,w is 3.6" lO -13 sec. The mol. wt. of 74 ooo was calculated from LANSINGANn KRAEMER'S17 equation applied to the results obtained in the equilibrium ultracentrifuge with both the original preparation of haemoprotein-55o from type I-ferrocytochrome b, and the preparation obtained from type II-ferrocytochrome b2 (see below). By moving-boundary electrophoresis in lactate-pyrophosphate buffer (I, about 0.6) and in lactate-KCl-imidazole buffer (I, about 0.2) only a single protein component was detected. At p H 6.2, 6.8 and 7.8, both the ascending and descending boundaries were symmetrical in both buffers for about 8 h. In one experiment in lactate pyrophosphate buffer at p H 6.8, after I I h some a s y m m e t r y of the boundary was apparent. From a plot of mobility versus pH, the protein was estimated to have zero mobility at p H 5.
The reaction of P C M S with cytochrome b2 Solutions of cytochrome b2 in o.i M sodium pyrophosphate, o.I mM E D T A (pH 6.8) were treated at 37 ° with either I or IO moles of PCMS/mole of haem; ferroand ferri-forms of both type I- and type II-cytochrome b~ were used, the former in the presence of 0.05 M sodium lactate. The changes in absorption spectra were followed with a recording spectrophotometer. Witlh type I- and type II-ferrocytochrome b2, the formation of cytochrome b 2ferrohaemoprotein-55o was indicated b y the progressive appearance of the typical spectrum of this compound. The rate was more rapid with the greater concentration of PCMB, but the same final extinction values at 55 ° m/z were obtained with both concentra~Lions. The final solutions were orange in colour and had a very strong r a v i n fluorescence; liberation of oxidized FMN was also indicated by the increase in absorption between 450 and 500 m#. Wit]a type I- and type II-ferricytochrome b2, the progress of the reaction was Biochim. Biophys. Acta, 96 (1965) 349-356
354
R . K . MORTON, K. SHEPLEY
indicated by the shift of the v-band from 416 m# (cytochrome b,) to 41o m# (haemoprotein-55o) and a decrease in the extinction. When no further change was apparent, the haemoprotein was reduced with sodium dithionite ; the spectrum of ferrohaemoprotein-55o was obtained. During the reaction of PCMS with ferricytochrome b~, flavin fluorescence appeared, as with the ferrocytochrome. With a solution of type I-ferrocytochrome b~ containing IO moles of PCMS/ mole of haem, the reaction at o ° was followed by observing the a- and r-bands of the haemoprotein with a low dispersion microspectroscope. After 3 days at o °, the aband had not changed from 557 mju, although the solution had a strong ravin fluorescence. On addition of acetone at o ° to this solution, the a-band rapidly migrated to 55 ° m/z, indicating formation of haemoprotein-55o. With only 0.5 mole of PCMS/ mole of haem at 37 °, spectroscopic observations indicated incomplete conversion of type I-ferrocytochrome b, to haemoprotein-55 o, since a-bands were clearly identifiable at 557 and 550 m# after several hours. A sample of type I-ferrocytochrome b~ in 0.3 M sodium lactate, 0.05 M sodium pyrophosphate, o.I mM EDTA (pH 6.8) was brought to 5 M urea and set aside for 48 h at 2 ° anaerobically. Then PCMS (19 mM final concentration) was added at 2 °. Spectroscopic observation indicated a progressive reaction to form haemoprotein-55o.
DISCUSSION
Cytochrome b, has protohaem as a prosthetic group but it differs considerably from other b-type cytochromes, especially in the magnitude of the coefficients of the principal extinction bands, (see MORTON18, Table V). This is not attributable to the presence of the FMN component, since the flavin-free derivative, cytochrome b~haemoprotein-557, has bands at almost identical wavelengths with similar elevated extinction coefficients4,5. The haemoprotein derivative described here was reported by MORTON4; it has a striking resemblance to heart-muscle cytochrome c in spectral and redox properties, and in reactivity with some enzymes. The prosthetic group is clearly not protohaem. The small amount of protohaem found in the first preparation may be attributed to some residual cytochrome b 2, the presence of which also accounts for the trace of ravin, the slight lactate dehydrogenase activity, and the slight asymmetry of the sedimenting boundary in the analytical ultracentrifuge. The failure to split from the protein with acetone-HCl, the cleavage with silver salt, and the identity of the product with that obtained from cytochrome c provide evidence that the prosthetic group of cytochrome b2-haemoprotein-55o is very similar to ferroporphyrin c and has at least one, and very probably two. thio-ether bonds to the protein. This conclusion is supported by the high reactivity of haemoprotein-55o with heart-muscle cytochrome c oxidase and with cytochrome c reductase, and by the marked change of E o' from about +o.12 V in cytochrome b, to about +0.28 V. In the absence of contrary evidence, it is tentatively concluded that the protohaem group of cytochrome b~ was converted to a ferroporphyrin c group, with thio-ether bonds to the protein. Extensive work has been directed toward the formation of thio-ether bonds from the vinyl side-chains of protoporphyrin and protohaem (see LEMBERG AND Biochim. Biophys. Acta, 96 (1965) 349-356
CYTOCHROMF b~-HAEMOPROTEIN-550
355
LEGGEI~). ZI~ILE2°, also ZEILE AND PIUTTI ~1, obtained compounds with an a-band at 55 o m# as a result of the vinyl side-chains of protohaem reacting with various amino acids, but thio-ether bonds were not formed. DEUL 2~, has described a reaction of 2,3 dimercaptopropanol with the vinyl group of protohaemoproteins in the presence of acetone. However, there appears to have been no previous account of a reaction forming a haemoprotein with thio-ether bonds to the polypeptide chain of the protein. Haemoprotein-55o has similar electrophoretic properties to cytochrome b2; it is apparently electrophoretically homogeneous and has zero mobility at about p H 5.o under the experimental conditions used. Horse-heart and yeast cytochromes c have iso-electric points of lO.65 (ref. 23) and 9.85 (ref. 24), respectively. Cytochrome b2 has I mole protohaem/82 ooo × g of protein2, 9. Since the extinction for 82 ooo × g of haemoprotein-55o is similar to that of 12 ooo x g of horseheart cytochrome c, it may be concluded that haemoprotein-55 o has I mole of haematin-c/82 ooo × g, and this is confirmed by the iron analysis. Whereas ferrocytochrom~: b2 has an S°2o,w value of 9.12 and a molecular weight from equilibrium sedimentatiLon of 177 300 (ref. 9), haemoprotein-55o has an S°2o,w value of 3.6o and a molecular weight from equilibrium sedimentation of 74 ooo. It is apparent, therefore, that the conversion of cytochrome b2 to haemoprotein-55o involves a reaction in which there is cleavage of the molecule. This reaction occurs in the presence of PCMS and of PCMB at 37 °, the rate increasing vcith temperature, and is almost instantaneous at 7 °0 or higher, but the protein is i hen rapidly denatured. It proceeds at a negligible rate at 0-2 ° except in the presence of acetone. The requirement of either an elevated temperature, or the presence ot! acetone at low temperature suggests that some unfolding of the protein, possibly involving rupture of hydrogen bonds, must precede the reaction with PCMS. This is supported b y the observation that haemoprotein-55 o is formed rapidly at o ° when PCMS is added to ferrocytochrome b~ in 5 M urea (p. 6). Although the specificity of the reaction has not been investigated, it is likely that other mercurials, such as methyl mercuric iodide, PCMB and possibly mercuric chloride react similarly to PCMS. Haemoprotein-55o was not formed by heating the enzyme in the presence of I M KCN, or of o.oi M 2,3-dimercaptopropanol. The reaction occurs with both ferri- and ferro-cytochrome b~. The shift of the maximum extinction in the ultraviolet region to about 257 m/~, and the very large increase in the extinction shows that the PCMS has combined with the protein and is still attached in haemoprotein-55o. Previously, ARMSTRONG et al. 25, have shown that there are four thiol groups/ haem of cytochrome b2 which react rapidly with PCMS. With 1-2 moles of PCMS/ mole of haem, the formation of oxygen-induced aggregates of the enzyme is prevented, ravin is displaced, and the dehydrogenase activity is substantially inhibited. This has been interpreted as indicating the presence of 1-2 thiol groups at the active centre of 1:he enzyme 2"~.Arsenite (io -2 M), Cu 2+ (lO-3 M), N-ethylmaleimide (IO-s M) all inhibit the enzyme substantially, supporting evidence for a thiol or thiol groups at the active centre, and this appears to be a thiol involved in binding of the ravin group. These observations are probably related to the formation of haemoprotein-55o, but are inadequate to explain it. There are, however, 18 half-cystine groups/mole of Biochim. Biophys. Acta, 96 (1965) 349-356
356
R . K . MORTON, K. SHEPLEY
haem in cytochrome b2 (ref. 26) and hence 14 half-cystine groups which apparently do not react with PCMS in the intact enzyme. Hence there are available a large number of potentially-reactive groups. Since the formation of haemoprotein-55o is accompanied by cleavage of the molecule of cytochrome b~, one possibility is that a disulphide bridge, close to the haem group, is cleaved and the thiols so formed attack the vinyl groups. CUNNINGHAM et al. ~v have reported a reaction of PCMB with disulphide bonds at elevated temperatures. Whatever the mechanism, it seems that the thiol (or disulphide) groups must initially be adjacent to the haem group of the enzyme. Further work will be directed toward elucidation of the mechanism of the reaction. Since cytochrome b~-haemoprotein-55o has covalent bonds between the polypeptide chain and the haem goup, this derivative should be suitable for studying the sequence of amino acids near the haem group. It is very unlikely that this sequence is modified from that present in cytochrome b, itself.
ACKNOWLEDGEMENTS
The assistance of Mr. R. CONNOLLYand the generous provision of substantial amounts of yeast by Messrs. Effront Yeast Co. Ltd., Melbourne, is gratefully acknowledged as is the grant-in-aid from the Nuffield Foundation. REFERENCES I C, A. APPLEBY AND R. K. MORTON, Nature, 173 (1954) 749. 2 C. A. APPLEBY AND R. K. MORTON, Biochem. J., 73 (1959) 539. 3 R. K. MORTON, J. McD. ARMSTRONG AND C. A. APPLEBY, Haematin Enzymes Syrup., Canberra, x959, P e r g a m o n Press, Oxford, 1961, p. 5Ol. 4 R. K. MORTON, Nature, 192 (1961) 727. 5 R. K. MORTON AND K. SHEPLEY, Biochem. Z., 338 (1963) 122. 6 C. A. APPLEBY AND R. K. MORTON, Biochem. J., 71 (1959) 492. 7 R. K. MORTON AND K. SItEPLEY, Biochem. J., 89 (1963) 257. 8 D. KEILIN AND E. F. HARTREE, Biochem. J., 39 (1945) 289. 9 J- McD. ARMSTRONG, J. H. COATES AND R. K. MORTON, Biochem. J. ,86 (1963) 136. IO D. KEILIN AND E. F. I-IARTREE, Biochem. J., 41 (1947) 5 °0. I I R . L . RINGLER, S. MINAKAMI AND T. P. SINGER, Biochem. Biophys. Res. Commun., 3 (196o) 417 . 12 E. ]3. SANDELL, Colorimetric Determination of Traces of Metals, Interscience, New York, 3rd edition, 1959, p. 54113 D. L. DRABKIN, J. Biol. Chem., 14o (1941) 387 . 14 O. H. LOWRY, N. J. ROSEBROUGH, A. L. FARR AND R . J . RANDALL, J. Biol, Chem., 193 (1951 ) 265. 15 J. McD. ARMSTRONg, J. H. COATES AND R. K. MORTON, Nature, 186 (196o) lO33. 16 R. HILL, in K. PAECH AND M. V. TRACEY, Modern Methods of Plant Analysis, Vol. I, SpringerVerlag, Berlin, 1956, p. 39317 W. D. LANSING AND E. O. KRAEMER, J. Am. Chem. Soc., 57 (1935) t369. 18 R. K. MORTON, Rev. Pure Appl. Chem., 8 (1958) 161. 19 R. LEMBERG AND J. W. LEGGE, Hematin Compounds and Bile Pigments, Interscience, N e w York, 1949, p. 2o4. 20 K. ZEILE, Z. Physiol. Chem., 207 (1932) 35. 21 K. ZEILE AND P. PIUTTI, Z. Physiol. Chem., 218 (1933) 52. 22 D. H. DEUL, Biochim. Biophys. Acta, 48 (1961) 242. 23 H. THEORELL AND A. ~kKESSON, J. Am. Chem. Sot., 63 (1941) 18o4. 24 J. McD. ARMSTRONG, J. H. COATES AND R. I~. MORTON, Haematin Enzymes Syrup., Canberra, 5959, P e r g a m o n Press, Oxford, 1961, p. 385 . 25 J. McD. ARMSTRONG, J. H. COATES AND R. K. MORTON, Biochem. J., 88 (1963) 266. 26 C. A. APPLEBY, R. K. MORTON AND D. H. SIMMONDS, Biochem. J., 75 (196o) 72. 27 L. W. CUNNINGHAM, B. J. NUENKE AND W. D. STRAYHORN, J. Biol. Chem., 228 (1957) 835.
Biochim. Biophys..4eta, 96 (1965) 349-356