SHORT COMMUNICATIONS 171

the sulfocompounds without further adaptation, considering that the utilization is strictly adaptive. Conversion of 35S from the labeled sulfonic acid substrates into cysteic acid of the insoluble fraction is presumed to involve biosynthesis of cysteine from the [3~S]sulfate produced during their metabolism. The results are consistent with the function of a sulfoglycolytic pathway 3 for bacterial degradation of sulfo- quinovose. The formation of sulfoacetate as product indicates the cell's preference for utilization of the derived triosephosphate as energy source. When sulfoacetate is the sole energy source, a further series of metabolic steps is required.

This work was supported by grants from the National Science Foundation (U.S.A.), the Institute of Chemistry, University of Brazil, and the National Council of Research of Brazil.

Department of Marine Biology, Scripps Institution of Oceanography, University of California, San Diego,

La Jolla, Calif. (U.S.A.)

HEBE L. MARTELLI*

A. A. BENSON

1 A. A. BENSON, H. DANIEL AND R. WISER, Proc. Natl. Acad. Sci. U.S., 45 (1959) 1582. R. A. FERRARI AND A. A. BENSON, Arch. Biochem. Biophys., 93 (1961) 185

3 A. A. BENSON AND I. SHIBUYA, Federation Proc., 20 (1961) 79. 4 T. YAGI AND A. A. BENSON, Biochim. Biophys. Aeta, 57 (1962) 6Ol. 5 A. A. BENSON, Advan. Lipid Res., I (1963) 387. 6 M. LEPAGE, H. DANIEL AND A. A. BENSON, J. Am. Chem. Soe., 83 (1961) 157. 7 H. DANIEL, M. MIYANO, R. O. MUMYCIA, T. YAGI, ~. LEPAGE, I. SHIBUYA AND A. A. BENSON,

J. Am. Chem. Soc., 83 (1962) 1765. S M. MIYANO AND A. A. BENSON, J. Am. Chem. Soc., 84 (1962) 59.

I. SHIBUYA AND A. A. BENSON, Nature, 192 (1961) 1186. 10 R. S. BREED; E. G. D. mURRAY AND N. R. SMITH, Bergey's Manual of Determinative Bacteriology,

The William and Wilkins Company, Baltimore, 1957. 11 S. WINOGRADSKY, Ann. Inst. Pasteur, 4 (189o) 257. 12 M. SmLo AND R. Y. STANIER, J. Sen. Microbiol., 16 (1957) 482. 1~ j . SPIZlZEN, ill S. P. COLOWICK AND 2{. O. I{APLAN, Methods of Enzymology, VoI. 5, Academic

Press, New York, I962, p. 122. 14 J. LEHMANN AND A. A. BENSON, J. Am. Chem. Soc., in the press. 15 A. A. BENSON, J. A. BAGSHAM, M. CALVIN, T. C. GOODALE, V. A. HAAS AND W. STEPKA, d r. A m.

Chem. Soc., 72 (195 o) 171o. 16 I. SHIBUYA, T. YAGI AND A. A. BENSON, Microalgae and Photosynthetic bacteria, Japan Soc. Plant

Physiologists, univ . Tokyo Press, 1963, p. 627.

Received June I l th , 1964

* Present address: Instituto de Qulmica, Universidade do Brasil, Rio de Janeiro (Brasil).

Biochim. Biophys. Aeta, 93 (I964) 169-171

sc 23o55

Enzymic formation of haems and other metalloporphyrins

Extracts that catalyse the incorporation of Fe ~+ into protoporphyrin have been pre- pared from liver mitochondria 1, avian erythrocytes~ and microorganisms 8. Differences in the metal specificity of such extracts have been reported. The liver mitochondrial extract formed Co-protoporphyrin as readily as Fe-protoporphyrin 4 but a chicken erythrocyte preparation did not incorporate 60Co2+ into protoporphyrin 5. It thus seemed possible that the capacity to form different metalloporphyrins varied between

Biochim. Biophys. Acta, 93 (1964) 171-173

~7 2 SHORT COMMUNICATIONS

species and might be related to a capacity to synthesise such metal- tetrapyrroie com- plexes as chlorophylls and vi tamin B1. ~ as well as the more commonly found haem pigments.

Extracts of avian erythrocytes 5, Rhodopseudomonas spheroides and T/~iobacitlus Xa, G have been used in our studies. Formation of haems, Co-porphyrins and Mn- porphyrins has been assayed by reduced minus oxidised difference spectra in alkatine pyridine 7, using the values for Co-porphyrins and Mn-porphyrins shown in Table ?i. The rate of incorporation of other metals was determined spectroscopically by meas- uring the rate of disappearance of Band IV of the porphyrin spectrum. The formation of a metalloporphyrin was confirmed from its characteristic absorption bands in the visible region. The extracts catalysed the incorporation of Fe 2+, Co 2+ and Zn ~+ (Table II). The previous failure to detect Co 2+ incorporation into protoporphyrin by erythrocytes 5 was probably due to the use of ethyl acetate to extract a°Co-proto- porphyrin formed during incubation. We have found Co-protoporphyrin to be in- soluble in this solvent. Previous failures to detect significant Zn l+ incorporation may be explained by the complexing action of the high concentrations of cysteine or GSH used in the incubation mixtures*, a.

No incorporation of Mg ~+ was detected, even in extracts of the chlorophyll, forming R. spheroides, when protoporphyrin or protoporphyrin monometi~yl ester was used, although Mg-protoporphyrin monomethyl ester is known to be a metabolite of R. spheroides s.

T A B L E i

DIFFERENCE SPECTRA OF REDUCED AND OXIDISED PYRIDINE COMPLEXES OF METALLOPORPHYRINS

Absorption maxima and minima (m#) zJ~mi-vi Complex a rain.

m~x~ . . . . ~im~m ~m~,im~m (m,,1 raM) a ~ i - s

C o - p r o t o p o r p h y r i n 554 533 512 9- -~ C o - d e u t e r o p o r p h y r i n 543 523 503 7-96 M n - m e s o p o r p h y r i n 584 57 ° 55 ° 3.85

T A B L E II

1VIETAL SPECIFICITY OF METALLOPORPHYRIN-FORMING ENZYMES FROM CHICKEN ERYTHROCYTES AND Thiobac i l lus X

I n c u b a t i o n s w e r e c a r r i e d o u t in T h u n b e r g t u b e s u n d e r N2, a-c 37 °. T h e i n c u b a t i o n m i x t u r e con- t a i n e d 200 m f z m o l e s of p o r p h y r i n , m e t a l s as i n d i c a t e d . 2oo # m o l e s p o t a s s i u m p h o s p h a t e buf fe r ( p H 8) a n d i . o m l e n z y m e e x t r a c t T h e f ina l v o l u m e was 4.2 ml . T h e r e a c t i o n w a s s t a r t e d b y t i p p i n g t h e m e t a l s a l t i n o. 4 m l a q u e o u s s o l u t i o n f r o m t h e s ide a r m of t h e T h u n b e r g tube . M n ~-. Mg 2+, Cd 2+, Ni ~+ a n d H g 2+ w e r e i ne f f e c t i ve az all c o n c e n t r a t i o n s used . All r a t e s w e r e c o r r e c t e d

for a low n o n - e n z y m i c , i n c o r p o r a t i o n of Z n 2+ o r Cu 2+.

Metalloporphyrin formed (m~mo~es/mg protein/h

Concentration Erythrocyte extract £hiobacilIus extract Metal ion (M)

Protoporphyrin Deuteroporphyrin Protoporphyrin Deuteroporphyrin

F e 2~ io 4 23.2 62.o 27.6 57 2 Co ~ 5" IO-5 ro.o 62.8 i i 7 62.0 Zn ~+ IO -~ 7 ° 75-3 i2 .2 63. 5

B i o c M m . B i o p h y s . Actc~, 93 (I964) I 7 I ~ I 7 3

SHORT COMMUNICATIONS 173

TABLE III

SEPARATION OF TWO HAEM-FORMING SYSTEMS FROM R. s])hgfoides

Incubat ions were carried out as described in Table II. The cells were treated for 2o rain in R a y t h e o n sonic osci l lator (25o VV, 1o kcycles) in o,1 M phosphate buffer (pH 7-4)- Crude fract ion was the

s u p e r n a t a n t of a 2o-min centr i fugat ion at 8000 × g.

Fraction Protohaem formed Deuterohaem formed Deuterohaem .formation (ml*moIes/h/mt extract) (m#moles/h/ml extract) Protohaem formation

Crude 99

S u p e r n a t a n t of 2 h centr i fugat ion at 70000 × g 4.8

Pellet f rom high-speed centr i fugat ion 55

18o 1.82

I21 25.2

38 0.69

After high-speed centrifugation of R. spheroides extracts the upper layer of super- natant had a very low protohaem-forming activity. However, deuterohaem was formed by this soluble extract (Table III) : act ivitywas restricted to deuteroporphyrin; mesoporphyrin, porphyrin c, 2,4-diethyl ester and porphyrin cytochrome c were not substrates although they too lack electrophilic side chains. This soluble enzyme from R. spheroides rapidly inserted Zn 2+ and Co 2+ into deuteroporphyrin but much less readily into protoporphyrin or other porphyrins.

These results suggest that the metalloporphyrin-forming enzymes may be specific to the porphyrin rather than the metal. This is supported by the finding that the incorporation of Co ~+ and Zn ~+ into protoporphyrin has a porphyrin specificity identi- cal w i t h that previously found a for Fe ~+ insertion. A Zn-protoporphyrin chelatase from chromatophore preparations of R. spheroides has been recently described 9. It was strongly inhibited by Co2+: our results suggest that this could be due to compe- tition between these two ions for a common enzyme. However , distinction can be made between the incorporation of Fe 2+ and of other metals; Fe 2+ incorporation is sensitive to - S H inhibitors at concentrations which are almost without effect on other metals. This may indicate that for insertion into porphyrins, Fe 2+ has to be stabilised by linkage to - S H groups, either on the enzyme or a coenzyme. Other metals do not have this requirement.

C.S.f.R.O., Division of Plant Industry, Canberra, A.C.T. (Australia)

ANNE JOHNSON

O. T. G. JONES*

1 C'. NISHIDA AND R. ~i'. LABBE, Biochim. Biophys. Ac)a, 31 (I959) 519. 2 R. C. XREUGER, J. MELNICK AND J. R. KLEIN, Arch. Biochem. B iophys . , 64 (I956) 302. a R. J. PORRA AS;D O. T. G. JONES, Biochem. f . , 87 (1963) 186.

R. F. LABBE AND N. HUBBARD, Biochim. Biophys. Acta, 52 (1961) 13o. 5 y . YONEYAMA, H. OHYAMA, Y. SUGITA AND H. YOSHIKAWA, Biochim. Biophys. Acta, 62 (1962)

261. 6 p. A. TRUDZNGER, Australian J. Biol. Sci., 17 (1964) 446. 7 R. J. PORRA AND O. T. C-. JONES, Biochem. J., 87 (1963) I8r . s O. T. C'. JONES, Biochem. J., 86 (1963) 429. 9 A. NEUBERGER AND G. H. TAIT, Biochem. J., 90 (1964) 607.

Received June 23rd, 1964

* Present address: Cheste~ford Park Research Station; Saffron Walden, Essex (c.reat Britain).

Biochim. Biophys. Acta, 93 (1964) 171-173