THE IRON AND PORPHYRIN METABOLISM OF MICROCOCCUS LYSODEIKTICUS*

BY P. M. TOWNSLEY AND J. B. NEILANDS

(From the Department of Biochemistry, University of California, Berkeley, California)

(Received for publication, April 20, 1956)

The elegant experiments of Shemin (1) as well as those of the schools of Rimington (2), Gibson et a.!. (3), and Granick (4) have done much to solve the over-all mechanism of porphyrin biosynthesis in the avian and mam- malian erythrocyte. However, most of this work has dealt with the origin of the porphyrin moiety of heme, and relatively little is as yet known of the mechanism of att,achment of the iron atom to the macrocyclic ring.

Working with Hemophilus influenzas, Granick and Gilder (5) observed that hemin or protoporphyrin IX could support growth of the organism. No metal-free porphyrin other than protoporphyrin was effective, although deutero-, meso-, and hematohemins were all active. These experiments established the “indirect evidence” for insertion of iron into protoporphyrin. Several years later Granick (6) found “direct evidence” for this reaction in extracts of chicken erythrocytes. However, in every case of porphyrin production by bacterial cultures grown at low iron levels, the extracted por- phyrin proved to be coproporphyrin rather than protoporphyrin. Al- though the list of organisms examined has remained small (Corynebuctetium diphtheriae (7, 8), Rhodospeudomonas spheroides (9)) Bacillus cereus (lo), and Ba&lus subtilis (ll), no exceptions to this rule were found.

The present investigation has dealt with the iron and porphyrin metab- olism of Micrococcus lysodeikticus. This organism offers several advantages as an experimental subject for such a study. In the first place, it is known to form very large amounts of the hemoprotein, catalase (12). Secondly, there was the possibility that under controlled iron nutrition the organism could be made to excrete free porphyrins, thus providing t,he opportunity for a study of the porphyrin metabolism as a function of iron supply. Fi- nally, since M. lysodeikticus is susceptible to lysis by lysozyme, the work with whole cells could be readily extended to soluble preparations.

The results reported in this paper show that, in either growing cultures or lysed cell preparations of M. lysodeikticua, an iron deficiency results in

* Abstracted from the doctoral dissertation of Philip McNair Townsley, Uni- versity of California, Berkeley, California. This work was supported in part by grant No. G-3993 from the Department of Health, Education, and Welfare of the National Institutes of Health, Public Health Service.

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696 METABOLISM OF M. LYSODEIKTICUS

the accumulation of coproporphyrin III. In addition, lysed cell prep- arations of this organism are capable of synthesizing coproheme from co- proporphyrinogen III and iron.

Methods

Materials-A culture of M. lysodeikticus and a highly purified prepara- tion of lysozyme were received from the Western Utilization Research Branch, Agricultural Research Service, United States Department of Agri- culture, Albany, California.

The liquid medium of Wessman et al. (13) was used, with three minor modifications; (a) the amino acid mixture was replaced with 5 gm. per liter of a casein acid hydrolysate, (b) the glucose content was increased to 10 gm. per liter, and (c) the trace elements were altered, both to account for the elements removed in demineralization and to provide metals which might be involved in porphyrin metabolism. The reagent grade trace elements were, per liter, Na&Io0,*2Hz0, 0.05 mg.; CuSOI.5Hz0, 0.39 mg.; MnCls*4HsO, 0.07 mg.; MgSOa.7Hz0 (recrystallized), 0.25 gm.; &SO,.- 7Hz0, 0.88 mg.; and NazBIOl. lOH~O,0.09 mg. Iron, as FeSOI.7H00, was added at a concentration calculated from an iron versus growth curve. The “low iron”’ media were prepared either by 8-hydroxyquinoline extraction (14) or by aluminum oxide-calcium oxide absorption (15). The inoculated cultures, in 200 ml. of medium, were incubated on a rotary shaker at 30” for 48 hours.

Lysis of M. lysodeikticus was accomplished by suspending the washed cells in a saline buffer solution (10 ml. of a 1 per cent NaCI solution plus 90 ml. of 0.15 M sodium phosphate, pH 6.8) and adding approximately 1 mg. of lysozyme per gm. of dry cells. The reaction mixture was incubated at 30°, and periodic measurements of the turbidity were made until lysis was considered to be complete.

The &aminolevulinic acid was synthesized by the method of Neuberger and Scott (16), except that the final product was crystallized from 95 per cent ethanol rather than from absolute methanol. The melting point was 151-152” (uncorrected) and the neutral equivalent was 167, as the hydro- chloride. The apparent ionization constants determined with the difunc- tional pH recorder (17) were 4.18 and 8.60 at 25“. All of these properties matched exactly those of an authentic sample of b-aminolevulinic acid hy- drochloride obtained from Dr. David Shemin.

Crystalline porphobilinogen was prepared enzymatically (3) from a-an& nolevulinic acid and isolated by heavy metal precipitation procedures (18).

1 “Low iron” media refer to those in which the iron concentration is such that porphyrin is excreted. “Adequate iron” media are those in which a slight excess of iron has been added over that required to prevent porphyrin excretion.

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P. M. TOWNSLEY AND J. B. NEILANDS 697

The substance darkened at 130-135” and decomposed at 170-175” (uncor- rected). The RF in butanol-acetic acid solvent was 0.51, when the chroma- togram was either sprayed with Ehrlich reagent or heated in acetic acid vapor.

Coproporphyrin III was prepared via the crystalline methyl ester (19) from either LV. Zysodeikticus or R. subtilis fermentations (11). Copropor- phyrinogen III was obtained by reduction of coproporphyrin III with so- dium amalgam (20). Authentic samples of protoporphyrin IX, copropor- phyrin III, coproporphyrin I, and uroporphyrin I were obtained from either C. H. Gray, F. MacDonald, or C. J. Watson.

Coprohemin was synthesized from coproporphyrin III (21), and proto- hemin was isolated from hog blood (22).

Procedures-Porphobilinogen was determined with the Ehrlich reagent (18), the crystallized compound being used as a standard. Heme com- pounds were extracted (23) and determined as their pyridine hemochromes (24).

Coproporphyrin, hematoporphyrin, protoporphyrin, and their metal chelates were isolated by extraction with ether at pH 3.5 (25). Uropor- phyrin was isolated either by adsorption on talc, followed by elution with 6 N NHdOH (26)) or by adsorption on calcium phosphate-calcium hydroxide, followed by dissolution of the adsorbent in dilute HCl (27). The specific isomer, uroporphyrin III, was extracted from solution at pH 3.1 with ethyl acetate (27). The isolated porphyrins were characterized by solvent solu- bility (25), fractionation with HCl solutions (25), paper chromatography of the free (28) and esterified compounds (29), column chromatography of the methyl esters (30), melting points (31), and absorption spectra of the methyl esters (25).

Porphyrins Excreted by Growing Cultures at Low Iron Levels

IdentiJication of Porphyrins-Cultivation of M. lysodeikticus in the “low iron” medium resulted in the production of suflicient porphyrin to color the medium a deep pink. Most of this material could be extracted into ether- acetic acid, and from this solvent it could subsequently be removed with 0.01 to 0.05 K HCl. Paper chromatographic analysis (28) revealed the presence of a single component with an RF identical with that of authentic coproporphyrin III. Chromatography of the methyl ester of the unknown porphyrin in a second solvent system (32) showed the substance to behave as coproporphyrin III. The methyl ester exhibited the characteristic co- proporphyrin III tetramethyl ester dimorphic melting point of 140’ and 157” (31). The absorption spectrum of the ester in dioxane gave principal maxima at 623, 568, 530, and 495 rnp. These figures are identical with

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698 METABOLISM OF M. LYSODEIKTICUS

those found by us for a sample of authentic coproporphyrin III. The Soret band maximum was at 399 rnp. The unknown porphyrin ester could not be separated from coproporphyrin III tetramethyl ester by column chromatography on either calcium carbonate or magnesium oxide. Thus, the porphyrin excreted by M. lysodeikticus has been demonstrated to be coproporphyrin III.

Although coproporphyrin was the only free porphyrin found in the growth medium, there was evidence that copper and zinc metalloporphy- rins were also present. For example, a non-fluorescent metalloporphyrin could be adsorbed on talc and eluted with acetic acid. The spectrum of this substance in ether-acetic acid revealed maxima at 561 (band I), 525 (band II), and 398 (Soret band) rnp, (I) > (II). A second metalloporphy- rin could be extracted from larger scale paper chromatograms. This com-

TABLE I Porphyrin Excretion by M. lgsodeikticua as Function of

Iron Added to Culture Medium

Iron added

y atoms*

8.85

1.79 1.43 0.89 0.18

* Per liter of medium.

Coproporphyrin III excreted

WlWlCS’

0.05 8.35 6.85 5.25

Very slight growth

Cell dry weight

1x5 1.96 1.77 1.87 0.04

pound was strongly fluorescent and had light absorption maxima at 563 (I), 543 (II), and 417 (Soret) rnp, (I) > (II). Thus the two metallopor- phyrins exhibited the known properties of the copper and zinc chelates of coproporphyrin, respectively.

Quantitative E$ect of Iron-At very low levels of iron there were little growth and consequently little porphyrin production. The data recorded in Table I show that the intermediate levels of iron were most effective in promoting porphyrin excretion. A point of considerable interest is the fact that, at certain levels, the iron acts in catalytic amounts to inhibit porphyrin accumulation. Thus, at the two highest levels of iron used (Table I), an increment of 7.1 y atoms of this element prevented the for- mation of 8.3 ccmoles of coproporphyrin III.

Prooh& of 6-Aminolevulinic Acid Metabolism by Cell Lysates

Porphyrinogena-The fact that cell lysates of M. lysodeikticus were quite transparent offered a unique opportunity for direct spectral analysis of

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P. M. TOWNSLEY AND J. B. NEILANDS 699

these preparations during porphyrin synthesis from d-aminolevulinic acid, When the amino acid was added to the lysates and incubation was carried out for 4 hours, under both aerobic and anaerobic conditions, practically no Soret band could be found. Similarly, the lysates did not show any fluorescence until after 4 hours, when a faint orange fluorescence appeared in the aerobic sample. Brief irradiation of these preparations with ultra- violet light caused the appearance of both fluorescence and a very intense Soret band. In addition, the almost colorless ether extracts of the lysates contained a substance which, on standing or irradiation, gave rise to rel- atively vast amounts of porphyrin.

Thus, from these data it is obvious that b-aminolevulinic acid actually gives rise to porphyrin derivatives which are non-fluorescent, without a Soret band, are soluble in ether, and readily yield porphyrin on standing or on exposure to ultraviolet light. The porphyrinogens are the only known substances which satisfy these properties.

The synthesis of substances behaving as porphyrinogens by cell lysates of M: lysodeikticus will be continued to be referred to as “porphyrin syn- thesis,” since the compounds were analyzed in the oxidized form.

Porphobilinogen-The first point of interest was the capacity of the two types of cell lysates, “low iron” and “adequate iron,” to synthesize the monopyrrole, porphobilinogen. In experiments which are not recorded in detail here, it was found that the amount of porphobilinogen present at any time in these preparations was independent of the gas phase. How- ever, some 20 per cent less porphobilinogen was formed per gm. of dry weight of the “adequate iron” lysates.

Porphyrins-Since porphobilinogen occurs as a transient compound be- tween &aminolevulinic acid and porphyrin, the lower levels of the mono- pyrrole present in the “adequate iron” lysate could have been attributed either to an inability to synthesize this compound or, by an enhanced ca- pacity to convert it to porphyrin or metalloporphyrin. In order to de- cide between these alternatives, the porphyrins present in the lysates were extracted, identified, and analyzed quantitatively. These data were pre- sented in Table II. Protoporphyrin was never found, even in trace amounts.

The quantity of coproporphyrin present (after 6 hours incubation) was a function of both the gas phase and the type of cell lysate. Thus, the greatest amount occurred in the “low iron” anaerobic preparation and the least amount in the “adequate iron” aerobic lysed cells. The presence of a small but significant fraction of difficultly extractable coproporphyrin should be noted. Once extracted from the organic solvent layer with 1.37 N HCl, this fraction exhibited all of the solubility and other properties of coproporphyrin III. This material was not identified, although, since it

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700 METABOLISM OF M. LYSODEIKTICUS

was fluorescent at all times, it may have existed in the organic solvent phase as the magnesium chelate of coproporphyrin III. All of the copro- porphyrin samples were contaminated with minor quantities of a material which behaved on paper chromatography as a pentacarboxylic acid por- phyrin.

Appreciable amounts of uroporphyrin were found. With the “low iron” samples this was mainly uroporphyrin III, but with “ adequate iron” sam- ples the isomer I occurred in higher concentration.

Efect of Added Iron-It was thus evident that some constituent of the

TABLE II

Porphyrins Extracted From “Low Iron” and “Adequate Iron” Lysed Cell Preparations of M. lysodeikticus with &Aminolevulinic Acid As Substrate

The reaction mixtures contained 0.60 gm. of dry cell weight and 56 pmoles of &aminolevulinic acid in a volume of 50 ml., pH 6.8. Incubation was carried out at 30’ for 6 hours on the slow rotary shaker in the dark. Control samples without added 6aminolevulinic acid did not yield detectable amounts of porphyrin.

Porphyrin extracted, ~~~~oles per gm. of dry lysed cell preparation

“Low iron” lysed cells “Adequate iron” lysed cells

Aerobic Anaerobic Aerobic Anaerobic

Coproporphyrin* 0.15 N HCl extract. . . . . . . . . . . 0.58 1.16 0.06 0.75 1.37 “ “ “ . . . . . . . 0.11 0.04 0.02 0.01 Total. . . . . 0.69 1.20 0.08 0.76

Uroporphyrin I. . . . . . . 0.08 0.04 0.57 0.28 I‘ III. . . . . . . 0.73 0.43 0.12 0.14

Total porphyrin. . . . . . 1.50 1.67 0.77 1.18

* Probably mainly isomer III.

“adequate iron” cells had a profound effect on the synthesis of copropor- phyrin. The simplest explanation was that this substance was either the iron atom itself or some derivative thereof. In order to test this hypothe- sis, a large patch of “low iron” lysate was prepared, one aliquot was used as a control, and iron was added to each of the other aliquots at varying concentration.

The results (Table III) show that it was possible to simulate the capacity of the “adequate iron” lysate to prevent coproporphyrin accumulation through the mere expedient of adding ferrous sulfate to the “low iron” lysate. As with the growing cells (Table I), the iron was able to act at certain concentrations in catalytic amounts to suppress coproporphyrin production.

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P. M. TOWNSLEY AND J. B. NEILANDS 701

Metabolism of Coproporphyrinogen III

E$ect of Iron-The results thus far were again open to two interpreta- tions. Either the iron was inhibiting the formation of both porphobilinogen and coproporphyrin or, conversely, the iron participated in a reaction lead- ing to the removal of these compounds. Coproporphyrin itself could be recovered unchanged when this substance and excess ferrous sulfate were added to “low iron” lysed cell preparations. Accordingly, an experiment was designed in which coproporphyrinogen III and various levels of fer- rous sulfate were added to the “low iron” cell lysates. The results, which

TABLE III Effect of Iron on Coproporphyrin E&actable from “Low Iron” Lysed Cell Preparation of M. lysodeikticua with &Aminolevulinic Acid As Substrate

The reaction mixtures contained 0.36 gm. of dry cell weight, 18.1 pmoles of 6- aminolevulinic acid, and FeSO,.7HnO at the stated concentrations in a final volume of 25 ml., pH 6.8. Incubation was carried out aerobically at 30” for 6 hours on the slow rotary shaker in the dark.

Iron added

- y atoms

nil 0.02 0.19 0.34 1.68

16.83 33.66

* Probably isomer III. t Per gm. of dry lysate.

T Coproporphyrin extracted*

0.15 N HCl extract

WM

0.38 0.32 0.15 0.12 0.10 0.07 0.08

1.37 N HCl extract

mm

0.09

0.09 0.04 0.02 0.02 0.01 0.01

are not given in details, revealed that the amount of coproporphyrinogen recovered as the corresponding porphyrin was inversely proportional to the added iron.

Eject of Inhibitors-In order to examine in more detail the mechanism of action of the iron in removing coproporphyrinogen III, a number of inhibitors were employed. The results (Table IV) illustrate that neither cyanide, malonate, nor mild heat was able to abolish the effect of iron in preventing coproporphyrin recovery. Autoclaving the lysate for 15 min- utes prior to the addition of the reagents inhibited by some 50 per cent.

Coproheme Synthesis-At this stage of the investigation it was noted that lysed cell preparations which had received both iron and coproporphy- rinogen had a distinct greenish color. Part of this color could be attributed

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702 METABOLISM OF M. LYSODEIKTICUS

to coproheme. The latter substance was extracted and identified by hemo- chrome formation and paper and column chromatography, with authentic coprohemin as a reference standard. The highest yields of coproheme were obtained from the preparations containing sodium cyanide at a level of 0.01 M (see Table V).

Stability of Variow Porphyrins and Hemes Added to Cell Lysates

Coproporphyrin III and protoporphyrin IX were found to be stable and virtually incapable of being reduced to their porphyrinogens or of accepting

TABLE IV E$ect of Iron and various Inhibitors on COpTOpOTphyTin Recovery from

“Low Iron” Lysed Cell Preparations of M. lysodeikticus with COpTOpOTphyTinOgen III As Substrate

The reaction mixtures contained 0.31 gm. of dry cell weight and 1.3 rmoles of coproporphyrinogen III in a volume of 25 ml., pH 6.8. Incubation was carried out aerobically at 25” for 4.5 hours in the dark. Individual flasks contained 5 pmoles of FeS04-7H20, 250 &moles of NaCN, or 1.25 mmoles of sodium malonate. Heat treatment consisted of exposure at 80” for 3 minutes.

Coproporphyrin extracted* (duplicate samples)

Without iron.. . . . . . With iron.. . . . . . . . . . . . . . . .

“ “ + cyanide.. . . . . . . . “ “ + malonate . . . I( “ + mild heat.. .

* Probably isomer III. t Per gm. of dry lysate.

0.15 N HCI extract 1.37 N HCI extract

pmo1cst rm01cst pma1ct rmld

3.59 3.64 0.07 0.15 2.61 2.61 0.03 0.03 2.27 2.29 0.05 0.04 2.78 2.78 0.03 0.03 2.40 0.06

iron in the presence of cell lysates. However, both the endogenous proto- heme of M. lysodeikticus and added protoheme were rapidly degraded by lysed cell preparations. Only traces of endogcnous protoheme remained in 4 to 6 hour-old lysates. Although the product of this reaction was not identified, it was probably bile pigment. In contrast to the instability of protoheme, added coprohemin could be recovered quantitatively from cell lysates.

Spontaneous Binding of Iron by Porphyrins and Their Precurwrs

Over the past several years repeated attempts have been made in this laboratory to insert iron into porphyrins by a chemical process carried out at neutral pH and room temperature. In none of these experiments, re-

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P. Mr. TOWNSLEY AND J. B. NEILANDS 703

ported in more detail elsewhere (33), have we found evidence for the spontaneous binding of ferrous or ferric ions by protoporphyrin, copro- porphyrinogen III, or during the macrocyclization of porphobilinogen to form uroporphyrinogen and uroporphyrin.

TABLE V Coproheme Synthesis by “Low Iron” Lysed Cell Preparations of M.

lysodeikticue with Coproporphyrinogen III As Substrate The reaction mixtures and conditions were identical with those described in

Table IV.

Coprqmphyrin extracted* Hemin extractedt

pmolcs~ rmm

Without iron.. . . . . . . . . . . . . . . . . . 3.72 Not detectable With iron.. . . . . . . . . . . . . . . . . . 2.64 0.03

‘I “ + cyanide.. . . . . . . . . 2.33 0.19

* Probably isomer III. Includes both 0.15 N HCI extract and 1.37 N HCI extract. t Calculated from the pyridine a-hemochrome band at 545 nq~ by use of the mo-

lecular extinction coefficient of pyridine protohemochrome at 557 rnp, 3.48 X 10’ (24). $ Per gm. of dry lysate.

DISCUSSION

The observation that substances exhibiting the properties of porphy- rinogens rather than porphyrins arise from &aminolevulinic acid is in ac- cord with both the current ideas of other investigators (2, 34) and the recent demonstration that uroporphyrinogen III gives rise to heme (35).

The simplest explanation for the data presented in Tables I to V would be that an iron deficiency results in a metabolic block immediately after the coproporphyrinogen III stage. The accumulation of the latter sub- stance would then give rise to the coproporphyrin III so commonly en- countered. However, it is doubtful whether coproheme itself lies on the direct metabolic pathway of heme synthesis, since it was recovered un- changed from the cell lysates. The iron-binding activity of porphyrins in which some or all of the methene bridges are reduced has not been eluci- dated. It should be pointed out that the presence of methylene bridges would destroy the planarity of the macrocyclic porphyrin ring, thus ex- posing the pyrrole nitrogen atoms for possible iron binding.

Irrespective of whether coproheme itself occurs on the pathway of heme synthesis, the fact remains that lysed cell preparations of M. lysodeikticus possess an iron insertion mechanism. The heat stability of this system raises the question of the enzymatic nature of the reaction. It is possible that porphyrins with at least some of their methene bridges reduced could form mixed chelate compounds with an iron donor which on intramolecular

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704 METABOLISM OF M. LYSODEIKTICUS

oxidation-reduction could release the iron to the porphyrin ring. Natu- rally occurring iron chelate compounds with a high affinity for ferric and a low affinity for ferrous ions are known (36).

There are at least two possible explanations for the low level of iron needed to remove coproporphyrin both in the growing culture and in the lysed cell preparations. Iron might be required for either the oxidative decarboxylation or the a,/3 unsaturation of the carboxyethyl side chains of coproporphyrinogen. On the other hand, at least part of the role of traces of iron could be explained on the basis of the demonstrated insta- bility of heme itself. Such a degradation reaction would lead to a recycling of iron back into the main metabolic stream.

The authors are indebted to C. H. Gray, F. MacDonald, D. Shemin, C. J. Watson, and H. L. Wolin for gifts of porphyrins, metabolites, and cultures.

SUMMARY

1. Growing cultures of Micrococcu.s lysodeikticus excreted coproporphyrin III when cultured at low levels of iron.

2. In certain regions of the curve “iron added” verSuS “porphyrin ex- creted,” the iron acted in less than stoichiometric amounts to prevent porphyrin accumulation.

3. Compounds exhibiting the properties of porphyrinogens, rather than porphyrins, were synthesized from &aminolevulinic acid by cell lysates of M. lysodeikticus.

4. The types and amounts of porphyrins formed by the lysed cells were a function of the initial iron level of the medium on which the organism had been cultivated.

5. A “low iron” (iron level low enough to cause porphyrin excretion) lysate gave more porphobilinogen, uroporphyrin, and coproporphyrin than an “adequate iron” (iron level high enough to prevent porphyrin excretion) lysate. Porphyrin formation was especially pronounced under anaerobic conditions.

6. The “low iron” lysed cell preparation failed to metabolize copro- porphyrinogen.

7. The disappearance of coproporphyrinogen III in the presence of iron could be correlated with the formation of coproheme. This reaction was insensitive to mild heat, but was partially destroyed by drastic heat treat- ment.

8. The spontaneous binding of iron by various porphyrins and their precursors was investigated under approximately physiological conditions. No incorporation of the iron was found.

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P. M. TOWNSLEY AND J. B. NEILANDS 705

9. The catalytic effect of iron in promoting the uptake of coproporphy- rinogen III could be explained on the assumption that the iron of degraded heme was fed back into the heme-synthesizing system.

BIBLIOGRAPHY

1. Shemin, D., in Graff, S., Essays in biochemistry, New York (1956). 2. Rimington, C., Endeavour, 14, 126 (1955). 3. Gibson, K. D., Neuberger, A., and Scott, J. J., Biochem. J., 61,618 (1955). 4. Granick, S., in Greenberg, D. M., Chemical pathways of metabolism, New York,

1 (1954). 5. Granick, S., and Gilder, H., J. Gen. Physiol., 30, 1 (1946). 6. Granick, S., Federation Proc., 13,219 (1954). 7. Pappenheimer, A. M., Jr., J. Biol. Chem., 167, 251 (1947). 8. Gray, C. H., and Holt, L. B., J. Biol. Chem., 169,235 (1947). 9. Lascelles, J., Biochem. J., 62, 78 (1956).

10. Schaeffer, P., Biochim. et biophys. acta, 9, 362 (1952). 11. Garibaldi, J. A., Dissertation, University of California (1956). 12. Herbert, D., and Pinsent, J., Biochem. J., 43, 193 (1948). 13. Wessman, G. E., Allen, L. P., and Werkman, C. H., J. Bact., 67,554 (1954). 14. Waring, W. S., and Werkman, C. H., J. Bact., 66,82 (1953). 15. Van Heyningen, W. E., and Gladstone, G. P., Brit. J. Exp. Path., 34,202 (1953). 16. Neuberger, A., and Scott, J. J., J. Chem. Sot., 1820 (1954). 17. Neilands, J. B., and Cannon, M. D., Anal. Chem., 27, 29 (1955). 18. Cookson, G. II., and Rimington, C., Nature, 171,875 (1953). 19. Granick, S., Bogorad, L., and Jaffe, H., J. Biol. Chem., 202,801 (1953). 20. Fischer, H., and Orth, II., The chemistry of the pyrroles, Leipzig (1937). 21. Vestling, C. S., J. BioZ. Chem., 136, 623 (1940). 22. Fischer, H., Org. Syntheses, 21, 53 (1941). 23. Schwartz, S., Federation PTOC., 14, 717 (1955). 24. Paul, K. G., Acta them. Stand., 7, 1284 (1953). 25. Lemberg, R., and Legge, J. W., Hematin compounds and bile pigments, New

York (1949). 26. Bogorad, L., and Granick, S., J. BioZ. Chem., 202,793 (1953). 27. Sveinsson, S. L., Rimington, C., and Barnes, H. D., Stand. J. CZin. and Lab.

Invest., 1,2 (1949). 28. Nicholas, R. E. H., and Rimington, C., Biochem. J., 48, 306 (1951). 29. Falk, J. E., and Benson, A., Biochem. J., 66, 101 (1953). 30. Nicholas, R. E. H., Biochem. J., 48, 309 (1951). 31. Jopo, E. M., and O’Brien, J. R. P., Biochem. J., 39,239 (1945). 32. Chu, T. C., and Chu, J.-H., J. BioZ. Chem., 212, 1 (1955). 33. Townsley, P. M., Dissertation, University of California (1956). 34. Shemin, D., Russell, C. S., and Abramsky, T., J. Biol. Chem., 216,613 (1955). 35. Xcve, R. A., Labbe, R. F., and Aldrich, R. A., J. Am. Chem. Sot., 78,691 (1956). 36. Neilands, J. B., J. Am. Chem. Sot., 74, 4846 (1952).

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P. M. Townsley and J. B. NeilandsLYSODEIKTICUS

METABOLISM OF MICROCOCCUS THE IRON AND PORPHYRIN

1957, 224:695-705.J. Biol. Chem. 

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