Studies on the formation of protoporphyrin IX by anaerobic bacteria

252

BIOCHIMICA ET BIOPHYSICA ACTA

BBA 26831

S T U D I E S ON T H E F O R M A T I O N O F P R O T O P O R P H Y R I N

IX BY ANAEROBIC

BACTERIA*

MASATAKA MORI** and SEIYO SANO*** The Department of Public Health. Faculty of Medicine, Kyoto University, A'yoto (Japan) (Received October 22nd, 1971)

SUMMARY The conversion of c o p r o p o r p h y r i n o g e n I I I to p r o t o p o r p h y r i n I X was demons t r a f e d in t h e e n z y m e o b t a i n e d from an obligate anaerobe, Chromatiurn D, o n l y when t h e a s s a y was c o n d u c t e d aerobically. A l t e r n a t i v e electron acceptors could not replace m o l e c u l a r 02. P r o t o p o r p h y r i n f o r m a t i o n could not be d e m o n s t r a t e d in the presence of A T P , MgSO4 a n d L-methionine u n d e r anaerobic conditions. P r o t o p o r p h y r i n I X f o r m a t i o n from c o p r o p o r p h y r i n o g e n I l I was not demons t r a t e d in a cell-free e x t r a c t of a n o t h e r obligate anaerobe, Desulfovibrio vulgaris, in either aerobic or a n a e r o b i c systems. The a d d i t i o n of several electron a c c e p t o r s a n d sulfate was ineffective. U r o p o r p h y r i n was f o r m e d from & a m i n o l e v u l i n i c acid b y the e x t r a c t , b u t not c o p r o p o r p h y r i n a n d p r o t o p o r p h y r i n . C o p r o p o r p h y r i n o g e n a s e of Chromatium D was purified 23 times. Purification i n v o l v e d F r e n c h press e x t r a c t i o n , u l t r a c e n t r i f u g a t i o n at p H 6.o, h e a t t r e a t m e n t at 60 °C a n d c h r o m a t o g r a p h y on D E A E - c e l l u l o s e . The p H o p t i m u m was 6. 4. The K,,~ for c o p r o p o r p h y r i n o g e n I I I was a b o u t 35/~M. Chelating agents such as o - p h e n a n t h r o l i n e a n d 0q~,'-dipyridyl d i d not i n h i b i t t h e e n z y m e a n d Fe 2+ d i d not s t i m u l a t e it. The enz y m e a c t i v i t y was s t r o n g l y i n h i b i t e d b y H g ~+ a n d A g +.

INTRODUCTION The conversion of c o p r o p o r p h y r i n o g e n I I I to p r o t o p o r p h y r i n I X involves the o x i d a t i v e d e c a r b o x y l a t i o n of two p r o p i o n a t e side chains to form v i n y l groups. The e n z y m e from m a m m a l i a n liver m i t o c h o n d r i a has been shown to have an absolute r e q u i r e m e n t for m o l e c u l a r 02, which could not be r e p l a c e d b y a l t e r n a t i v e electron a c c e p t o r s 1,2. Sano 3 showed t h a t s y n t h e s i z e d 2,4-bis-(C?-hydroxypropionic acid)* This work forms part of a thesis submitted for the degree of Ph.D. to Kyoto University by Masataka Mori. **Present address: Department of Biochemistry, Chiba University School of Medicine, Chiba, Japan. *** To whom reprint requests should be directed.

Biochim. Biophys. Acta, 264 (1972) 252-262

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253

deuteroporphyrinogen IX was converted to protoporphyrin IX by a mitochondrial enzyme, suggesting that this hydroxyl compound resulting from an oxygenase-type reaction is dehydrated and decarboxylated to protoporphyrin IX. However, a similar oxygen-requiring mechanism for this oxidative conversion seems unlikely in obligate anaerobes, since these bacteria, e.g. Chromatium and Desulfovibrio, can synthesize c-type cytochromes or bacteriochlorophylls during growth under strictly anaerobic conditions. In these bacteria, it seems likely that an alternative mechanism of protoporphyrin formation not involving molecular 02 is employed. Despite wide interest in this problem~, 5, the enzyme had not been detected in cell-free extracts of both aerobically and anaerobically grown microorganisms until recently. Mori and Sano% using a cell-free extract of the obligately anaerobic photosynthetic bacterium, Chromatium, demonstrated that protoporphyrin IX was not formed from coproporphyrinogen III under anaerobic conditions, but unexpectedly formed under aerobic conditions. Several alternative electron acceptors could not replace the oxygen requirement. Recently, Tait 7 showed that an extract from Rhodopseudomonas spheroides, grown semi-anaerobically in the light, converted copropor~ phyrinogen III to protoporphyrin IX when incubated anaerobically in the dark with Mg 2+, ATP and L-methionine. In contrast, Jacobs et al 8, using extracts of anaerobically grown Pseudomonas denitrificans and Escherichia coli, showed that protoporphyrin accumulation was demonstrated only if the assay was conducted aerobically. Supplements of ATP, Mg 2+ and methionine had no effect on anaerobic formation of protoporphyrin in their system. The present paper deals with further studies on the formation of protoporphyrin IX from coproporphyrinogen III by cell-free extracts of Chromatium D and Desulfovibrio vulgaris. This paper also describes the partial purification of coproporphyrinogenase from Chrom~tium D and some of its properties which may shed some light on the understanding of the mechanism of protoporphyrin IX formation in anaerobic bacteria. EXPERIMENTAL PROCEDURE

Bacterial cultures Chromatium strain D was supplied by Dr S. Morita, University of Tokyo. It was cultured essentially as described by Morita et al. 9, except that the medium contained in % (w/v), NaC1, I.O; CaC12.2H20, 0.007; FeC13.6H20, o.ooo5 and no FeSO4.7H20. After the growth period (40-48 h), the cultures were chilled to 4 °C, and the cells were harvested by centrifugation and washed with cold 1% NaC1 solution previously bubbled through with N2. Cells were kept frozen at --20 °C unless used immediately. Desulfovibrio vulgaris was supplied by Prof. H. Ishimoto of Hokkaido University. It was cultured anaerobically for 2 days at 37 ° C in a medium modified from Ishimoto et al. ~°, having the following composition (%, w/v); peptone, 0.3; beef extract, o.I; calcium lactate, 0.3; MgS04.7H20, o.15; Na2SO~, o.15; KH~P04, o.oi. Porphyrins and porphyrinogens Coproporphyrin III and protoporphyrin IX were prepared by the method previously described 1. Uroporphyrin III was a gift of Prof. C. Rimington, University College Hospital Medical School, London, and uroporphyrin I from Dr T. K. With, Svendborg Country Hospital, Denmark. Biochim. Biophys. Acta, 264 (I972) 252-262

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M. MORI, S. SANO

Coproporphyrinogen I I I was prepared from coproporphyrin I I I solution by reduction with both sodium amalgam 1 and NaBH4. The latter method was as follows. Under N2 in dim light, 2o mg of NaBH4 were added to o.o5 ml of 1.6 mM coproporphyrin I I I in o.3 ml of i M Tris-HC1 buffer (pH 7.o), resulting in a completely colorless solution, cooled in an ice bath, neutralized by addition of o.o2 ml of acetic acid. This was used for one assay tube.

Other materials Chemicals were obtained as follows : DEAE-cellulose, from Serva Entwicklungslabor; ATP, &aminolevulinic acid and Tris, from Sigma Chemical Company; Sadenosylmethionine, from Boehringer and Soehne G m b H Mannheim. All other reagents were of analytical grade. Spinach ferredoxin was a gift of Dr T. Horio, Osaka University, and crystalline metapyrocatechase was a gift of Drs M. Nozaki and O. Hayaishi of Kyoto University.

Determination of 02 content in culture medium by metapyrocatcchase The oxygen content in the culture medium of Chromatium D was determined principally according to the method of Nozaki et al. 11. A 3o-ml test tube filled with freshly prepared culture medium containing 3 mM catechol and a small glass bead was stoppered by a rubber stopper without air space. After standing for 3 h at 25 °C, o.I ml of crystalline metapyrocatechase solution (IOO/~g protein) was added by injection through the rubber stopper and mixed. After 4 h incubation at 25 °C, a 5-ml aliquot was poured into 2 ml of 0.2 M/HC1 under N2, the protein was removed by centrifugation and the supernatant was neutralized to exactly pH 7-5. The product formed was determined spectrophotometrically at 375 nm (eM=224ooo).

Preparation of Chromatium and Desulfovibrio crude extract Chromatium crude extract was prepared as described previously 6, except that the pH of the Tris-HC1 buffer was 8.0. For anaerobic incubation it was used immediately. It could be stored for several days at --20 °C for further purification. Desulfovibrio crude extract was prepared by the same procedure as that of Chromatium crude extract, except that 2-mercaptoethanol was omitted.

Separation of porphyrins from reaction mixture with 6-aminolevulinic acid Uroporphyrin was extracted and determined according to the method of Falk '2. Uroporphyrin isomers were characterized by the method of Edmondson and Schwartz (ref. 13). Coproporphyrin and protoporphyrin were determined by the method of Sano and Granick 1.

Assay of coproporphyrinogenase activity Coproporphyrinogenase activity was measured essentially by the method of Sano and Granick 1. The aerobic reaction mixture contained o. I M potassium phosphate buffer, p H 6.5, 60-80 nmoles of coproporphyrinogen I I I (prepared both by sodium amalgam and NaBH4), o.oi M sodium diethyldithiocarbamate and enzyme preparation in a total volume of 4 ml. The amount of coproporphyrinogen I I I used was determined in every experiment by immediately oxidizing it with aqueous I2 to eoproporphyrin I I I . Anaerobic incubation with crude extract was carried out as follows. To conical

Biochim. Biophys. Acta, 264 (1972) 252-262

PROTOPORPHYRIN FORMATION BY ANAEROBIC BACTERIA

255

3o-ml test tubes flushed with N~, were added o.5-2.o ml of bacterial crude extract, 60-80 nmoles of coproporphyrinogen III, prepared both by sodium amalgam and NaBH4, o.I M potassium phosphate buffer (pH 6.5) or Tris-HC1 buffer (pH 8.o), IO mM sodium succinate, other compound and water saturated with N,~ to 2-4 ml. N~ was bubbled vigorously over the surface and tubes were stoppered. In some anaerobic experiments, reactions were performed under N~ in Thunberg tubes after two cycles of freezing, evacuation and flushing with N2. Protoporphyrin was characterized on the basis of spectral properties, paper chromatography and the rate of conversion into mesoporphyrin IX by catalytic hydrogenation 14-1~. Enzyme unit One unit of coproporphyrinogenase was defined as the amount of enzyme that catalyzed the formation of i nmole of protoporphyrin IX from coproporphyrinogen III under standard conditions. The specific activity was the units of coproporphyrinogenase per mg of protein. Protein was determined by the method of Lowry et al. 1~. RESULTS

02 content in culture medium of Chromatium The 02 concentration, determined as described in Experimental Procedure, was calculated to be less than o.5 #M. When the reaction mixture was opened to the atmosphere, the yellow color of the product promptly appeared and the reaction proceeded until the substrate catechol was consumed. When the reaction was carried out in o.i M potassium phosphate buffer, pH 7-5, equilibrated with air at 25 °C, the concentration of 02 was determined to be 0.246 mM, indicating that the 02 dissolved in buffer solution at 25 °C was completely consumed by metapyrocatechase and catechol. Chromatium was found to grow in the culture medium containing metapyrocatechase and catechol at the same rate as in the usual culture medium. Examination of Chromatium crude extract for ability to convert coproporphyrinogen I I I into protoporphyrin I X under both anaerobic and aerobic conditions As reported earlierL no protoporphyrin was formed from coproporphyrinogen III under anaerobic conditions in the presence of a variety of electron acceptors such as NAD +, NADP +, FAD, FMN (5/~moles, each), methylene blue, phenazine methosulfate, 2,6-dichlorophenol indophenol, 2,3,5-triphenyltetrazorium chloride, MnO2, potassium ferricyanide, chloranil (IO #moles, each), horse heart cytochrome c (7.3 /,moles), sodium tetrathionate and sodium fumarate (IOO #moles, each) (Table I). No effect was shown by the addition of boiled extracts, coenzyme A (1. 4 #moles) plus ATP (3.o #moles), cysteine (4/,moles, 40/,moles), glutathione (4 #moles, 4o/,moles), spinach ferredoxin (approx. 3 rag) and Chromatium ferredoxin (approx. 20 rag). No protoporphyrin was observed, even when the extract was supplemented with ATP, MgSO4, methionine or ATP, MgSO4, S-adenosylmethionine and all of the added coproporphyrinogen was recovered as coproporphyrin (Table I). Whether or not coproporphyrinogen was prepared by reduction with either sodium amalgam or sodium borohydride, similar results were obtained under both anaerobic and aerobic conditions. Additions of a number of sulfur compounds and amino acids were also Biochim. Biophys. Acta, 264 (1972) 252-262

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M. MORI, S. SANO

TABLE I EFFECT OF SUPPLEMENTS ON PNOTOPORPHYRIN FORMATION BY Chromatium CRUDE EXTRACT UNDER ANAEROBIC CONDITIONS I n c u b a t i o n m i x t u r e contained o.i M p h o s p h a t e buffer, p H 6. 5 or Tris-HC1 buffer, p H 7.4 or 8.0, c o p r o p o r p h y r i nogen [ I I , crude e x t r a c t (30-43 mg protein), 20 #moles of s o d i u m succinate (anaerobic conditions) and indicated s u p p l e m e n t s in a total volume of 2.0 ml. S u p p l e m e n t s are: NAD+, N A D P +, F A D (5 /~moles, each), methylene blue, phenazine methosulfate, 2,6-dichlorophenol indophenol, sodium Ierricyanide (IO/,moles, each), horse h e a r t ferricytochrome c (7.3 /mloles), A T P (io/~moles), MgSO~ (50/~nloles), L-methionine and S-adenosylmethionine (5 /~moles, each) and FeSO 4 (2o/mloles). (A) C o p r o p o r p h y r i n o g e n p r e p a r e d by sodium a m a l g a m . (B) Coprop o r p h y r i n o g e n p r e p a r e d b y NaBHa.

Group Incubation condition p H

Time (rain)

Addition

Coproporphyrinogen added

Porphyrin recovered (nmoles) Copro Proto

62.8 62.8 33.8 80.0 80.0 80.0 80.0 80.0 80.0

21.6 28.6 26.0 28.o 69.6 74.0 77.0 70..5 70.4 80.0

34.2 17. 9 o o o o o o o

80.0 80.0 33.8 64.0 64.0

60.0 57.0 28.6 31.8 61.8-62.o 61.8

o o o o o

62.8 62.8 57-o 57.o

34.2 36.9 54.5-56.5 48.5-53.o

25.o 8. 7 o o

(nmotes)

A

Aerobic

B

6.5* 8.0* Anaerobic 6. 5 or 8.0 7.4** 7-4** 7-4** 7.4** 7.4** 7.4**

4° 4° 18o 12o 12o 12o 18o 18o 18o

7.4** 7.4** 6. 5 or 8.0 6. 5 or 8.0 8.0

18o 18o 18o 12o 12o

None None None N A D + (--0,32 m V * * * ) N A D P + ( 0.32 nlV***) F A D (--o.18 m V * * * ) Methylene blue ( + o . o i m V * * * ) PheBazine methosulfate ( + 0 . 0 8 m V * * * ) 2.6-Dichlorophenol indophenol (+0.22 mV***) Ferricytochrome c (+0.22 mV***) Sodium ferricyanide ( + 0 . 4 9 m V * * * ) ATP, Mg 2+, methionine ATP, Mg 2+, S - a d e n o s y h n e t h i o n i n e ATP, Mg 2+, S-adenosyllnethionine, Fe z+

6.5 8.o Anaerobic 6. 5 or 8.o 6. 5 or 8.o

4° 4° 12o 12o

None None ATP, Mg 2+, nlethionine ATP, Mg 2+, S-adenosylmethionine

Aerobic

* Crude e x t r a c t (84 mg protein) incubated in total volume of 4.o nil. Crude e x t r a c t (6o mg protein) incubated in the absence of sodium succinate in a total volume of 4.0 ml. *** Redox potential at p H 7.o. **

ineffective in anaerobic protoporphyrin formation. Those tested were DL-methionine, S-methylmethionine iodide, DL-acetylmethionine, dimethyl sulfoxide, diphenyl sulfone, L-serine, L-threonine, L-tyrosine, L-histidine and L-arginine (2/,moles, each). However, the extract converted coproporphyrinogen to protoporphyrin when assayed aerobically at pH 6. 5 or 8.0 (Table I).

A erobic and anaerobic incubation of Desulfovibrio crude extract with coproporphyrinogen III No protoporphyrin formation was demonstrated when Desulfovibrio crude extract (approx. 40 mg protein) was incubated with coproporphyrinogen III (60-80 nmoles) under both aerobic and anaerobic conditions. Addition of electron acceptors such as NAD +, NADP +, FAD (2/,moles, each), methylene blue, 2,6-dichlorophenol indophenol, phenazine methosulfate and potassium ferricyanide (I/,mole, each) under anaerobic conditions had no effect. The presence of Na~S04, Na2SO3 or Na2S20~ (20 /,moles, each) or ATP, MgSOa plus L-methionine (IO /,moles, 5 ° /,moles, 5 /~moles, respectively) also did not allow protoporphyrin formation. Bioehim. Biophys. Acta, 264 (1972) 252-262

PROTOPORPHYRIN TABLE

FORMATION BY ANAEROBIC BACTERIA

257

II

PORPHYRIN FORMATION FROM ~-AMINOLEVULINIC ACID BY Desulfovibrio CRUDE EXTRACT

Desulfovibrio c r u d e e x t r a c t (76 m g p r o t e i n ) i n c u b a t e d a t 37 °C i n o . i M T r i s - H C 1 b u f f e r , p H 7-4, i n a t o t a l v o l u m e of 4 . 0 m l w i t h b - a m i n o l e v u l i n i c a c i d . Incubation condition

Incubation time (rain)

d-Aminolevulinic acid added (nmoles)

Porphyrins (nmoles) Uro Copro Proto

Aerobic

4° 240 40 24o

745 745 745 745

73.1 344 73.4 368

Anaerobic

× × × ×

8 8 8 8

o o o o

o o o o

Porphyrin formation from &aminolevulinic acid by Desulfovibrio crude extract The crude extract contained no detectable amount of endogenous porphyrins. Whether incubation was carried out aerobically or anaerobically, formation of uroprophyrin from &aminolevulinic acid proceeded with time, and after 4 h about tmlf of the added 6-aminolevulinic acid was converted to uroporphyrin. However, no detectable coproporphyrin and protoporphyrin was observed (Table II). When the assay was carried out in freshly prepared culture medium, almost the same results were obtained. Formed uroporphyrin contained both Isomer I I I and I and the ratio of I I I to I varied from one experiment to another ranging from 80 : 20 55 : 45. The ratio was also influenced by incubation time, decreasing with longer incubation, probably due to the inactivation of uroporphyrinogen I I I cosynthetase during incubation. When uroporphyrinogen I I I was incubated with freshly prepared crude extract under both aerobic and anaerobic conditions, coproporphyrinogen I I I was not formed. Uroporphyrinogen I I I was recovered as uroporphyrin in a yield of 70-800/0 . These results show that Desulfovibrio crude extract had very little activity of uroporphyrinogen decarboxylase.

Further purification of coproporphyrinogenase of Chromatium D Subsequent operations were performed at 0- 4 °C unless otherwise indicated. Ultracentrifugation of crude extract at pH 6.o. Crude extract (82.0 ml) prepared as described in Experimental Procedure was adjusted to pH 6.0 by the dropwise addition of I M HC1 and was centrifuged at 105000 × g for 60 rain. 70-8o% of the enzymatic activity of the crude extract was found in the clear brown supernatant and no activity was found in the dark red precipitate. When the ultracentrifugation was performed at pH 7.0 or above, it was difficult to remove all of chromatophores and other particles, resulting in a slightly turbid, reddish-brown supernatant. Heat treatment. The supernatant from ultracentrifugation (66.o ml) was adjusted to pH 8.0 by o.I M NaOH and heated at 60 °C for io rain. Coagulated proteins were centrifuged off at 15 ooo × g for 20 rain and discarded. DEAE-cellulose column chromatograph),. The supernatant enzyme from the proceeding step (57.0 ml) was diluted with 2 vol. of cold water and chromatographed on a DEAE-cellulose column (2 c m × 2 7 cm) previously equilibrated with o.oi M Tris-HC1 buffer, pH 8.0. It was subjected to linear gradient elution with the same buffer containing NaC1 of between o and 0.35 M (400 ml) at a flow rate of 40 ml per h; succesive fractions of 4 ml were collected. The enzyme was eluted in a single peak in approximately 0.25 M NaC1 elution (Fig. I). The enzyme solution (32 ml) with the Biochim. Biophys. Acta, 2 6 4 (1972) 2 5 2 - 2 6 2

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M. MORI, S. SANO

highest activities was concentrated by precipitation with 12.5 g of solid (NH4)2 SO4 (6O~o saturation) and the precipitated fraction was collected by centrifugation and redissolved in 5.0 ml of 0.02 M Tris-HC1 buffer, pH 8.0. A clear, pale yellow-brown preparation was obtained. When it was dialyzed against 0.02 M Tris-HC1 buffer, pH 8.0, no change in enzymic activity was observed. Table III summarizes a typical result of the purification procedure.

Properties of partially purified coproporphyrinogenase under anaerobic conditions of assay Partially purified enzyme (DEAE-cellullose step) was incubated with 66-75 nmoles of coproporphyrinogen III anaerobically for 2 11 in the absence and presence of electron acceptors, such as FAD (5 #moles and 0.5 #mole), FMN (5 pmoles), NAD +, NADP +, methylene blue, 2,6-dichlorophenol indophenol, phenazine methosulfate and potassium ferricyanide (z #moles, each). No protoporphyrin IX was formed.

Properties of partially purified coproporphyrinogenase under aerobic conditions of assay Effect of pH. The pH optimum for coproporphyrinogenase was 6. 4 in o.I M potassium phosphate buffer. The activity was equal in o.I M Tris-HC1 buffer and sodium citrate buffer. Effect of incubation time. Under the standard conditions described, coproporphyrinogenase activity was approximately linear with time up to 4 ° min, and decreased with longer incubation periods. Enzyme inactivation and decrease of substrate by autoxidation and by conversion to protoporphyrin probably occurred with a longer incubation time, resulting in decreased enzyme activity.

F
2O

>-

z '

15 > I.--

1.0 rn

s~ N 'o,,z, 00

20

40

60

80

100

FRACTION NUMBER Fig. I. C h r o m a t o g r a p h y of Chromatiumcoproporphyrinogenase on a D E A E - c e l l u l o s e c o l u m n . See t h e t e x t for details. © - - © , a b s o r b a n c e a t 28o n m ; O - - e , c o p r o p o r p h y r i n o g e n a s e a c t i v i t y . TABLE III PURIFICATION

OF

COPROPORPHYRINOGENASE

Total unit Cr ude e x t r a c t t74o Ultracentrifugation 13oo H e a t t r e a t m e n t a t 60 °C 694 D E A E - c e l l u l o s e c h r o m a t o g r a p h y 378

Biochim. Biophys. Acta, 264 (I972) 252-262

OF

Chromatium D Total protein (rag)

Specific activity (units~rag protein)

Yield (%)

335 ° lO82 314 31.o

o.52 1.2o 2.21 12.2

ioo 74.8 39.8 21. 7

259

PROTOPORPHYRIN FORMATION BY ANAEROBIC BACTERIA

18 . . . .

A

o taJ

or. o LL 0 Z "1"

a. 5 0 0.. 0

f

25 50 75 I O0 COPROPORPHYRINOGEN ( ,uM )

i

lO0

o

50

j

0 20 40 6 0 8 0 1 / COPROPORPHYRINOGEN ( mM )

Fig. 2. (A) Effect of c o p r o p o r p h y r i n o g e n concentration on c o p r o p o r p h y r i n o g e n a s e activity. The s t a n d a r d assay s y s t e m was used. The reaction m i x t u r e contained partially purified enzyme (1.2 m g protein) and c o p r o p o r p h y r i n o g e n in various concentrations as indicated. (B) L i n e w e a v e r - B u r k plots for c o p r o p o r p h y r i n o g e n I I I . TABLE IV EFFECT OF VARIOUS COMPOUNDS ON AEROBIC COPROPORPHYRINOGENASE ACTIVITY OF Chromatium The assay s y s t e m contained o . I M p h o s p h a t e buffer, p H 6.5 (Group D, o.iM Tris-HC1 buffer, p H 7.o), 1.2-2. 5 m g of purified e n z y m e p r e p a r a t i o n , 60-70 nmoles of c o p r o p o r p h y r i n o g e n I n a n d c o m p o u n d s as indicated, in a t o t a l v o l u m e of 4 ml. D i e t h y l d i t h i o c a r b a m a t e was omitted. I n c u b a t i o n was carried o u t u n d e r s t a n d a r d conditions.

Group

Compound

Concentration (raM)

Control A

Cysteiue

A ctivity (%) I oo

Porphyrin recovery ol (/o) 82-95

2-Mercaptoethanol

io i io I i

B

Iodoacetamide N-Ethylmaleimide p-Chloromercuribenzoate

io Io o.o5

89 13 I 15

i o1 18 87

C

o-Phenanthroline ~, ~'-Dipyridyl 8-Hydroxyquinoline EDTA Tiron Sodium d i e t h y l d i t h i o c a r b a m a t e NaCN H y d r o x y l a m i n e • HC1

io io I io I io IO io

123 98 lO5 lO 3 93 169 78 75

79 75 93 85 72 94 69 83

D

FeSO 4

I o.i I o.i I i I

88 96 IOi 45 93 123 92

64 83 79 32 76 72 96

13 54 33

82 96 72

GSH

FeCI 3 CuC12 MgClz CaC12 ZnC12 E

HgC12

85 94 75 84 I oo

76 92 81 76 91

AgC1

I o.i I

F

Quinacline • HC1 Riboflavin

i o.o2

72 108

81 85

G

SKF-525A

o.i

Io 7

81

Biochim. Biophys. Mcta, 264 (I972)~252-262

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Effect oJ enzyme concentration. Coproporphyrinogenase activity was approximately proportional to the concentration of enzyme protein up to 2 rag. Since less than 2.5 mg protein was used in the assay, this upper limit of protein concentration was not exceeded. Effect of substrate concentration and the K,n value. The actual substrate concentration was not known because of autoxidation of coproporphyrinogen during incubation; the apparent Km value was about 35 #M (Fig. 2). This is in good agreement with values reported for enzyme preparations from other sourcesl,ls, 19. Effect of various substances on coproporphyrinogenase. The effect of various compounds on the purified enzyme preparation is shown in Table IV. Thiols and sulfhydryl reagents had little effect on the activity (Table IV, Groups A and B). In the presence of N-ethylmaleimide, porphyrin destruction occurred. Diethyldithiocarbamate stimulated the activity 1.6-2.o-fold (Table IV, Group C). This compound enhanced the activity of purified preparations (DEAE-cellulose fraction, heat treatment fraction) but had little effect on crude extract and IO5OOO×g supernatant. Other metal chelators were without appreciable effect, in contrast to the data from beef liver mitochondrial enzyme 1. Ferrous ion, at I mM or o.i mM, did not stimulate but rather slightly inhibited the formation of protoporphyrin (Table IV, Group D). Porphyrin destruction or copper-coproporphyrin formation occurred in the presence of cupric ion. The enzyme was inhibited approx. 9 ° and 50% by Hg 2+ at I and o.I raM, and approx. 70% by I mM Ag +, respectively (Table IV, Group E). The autoxidation of coproporphyrinogen I I I was not observed in the presence of Hg ~+ or Ag+. SKF-525A, an inhibitor of some hydroxylation reactions, showed no inhibition at I, o.I and o.oi raM. DISCUSSION Molecular O2 is essential for the conversion of coproporphyrinogen I I I to protoporphyrin I X by mammalian liver mitochondrial enzymes, and alternative electron acceptors can not replace molecular 0.,1, 2. This finding is most interesting and puzzling in the light of the distribution of protoporphyrin in advanced living organisms. Hemoproteins are not found in strictly anaerobic clostridia, in the microaerophilic laetobacilli or in the facultatively anaerobic streptococci or pneumococci 2°. On the other hand, certain anaerobic sulfate-reducing bacteria such as Desulfovibrio vulgaris and Desulfovibrio desulfuricans contain m a n y c-type cytochromesl°, 21. The strictly anaerobic green and red sulfur bacteria are rich in cytochromes and bacteriochlorophylls (ref. 22, 23). Facultatively anaerobic nonsulfur purple bacteria and some nonphotosynthetic facultative anaerobes can also synthesize cytochromes and bacteriochlorophylls when grown anaerobically 24-27. In such anaerobic bacteria, protoporphyrin m a y be formed by an alternative mechanism not involving molecular 02. The hypothesis is supported by 02 content in the culture medium of Chromatium. It was found to be less than 0. 5/zM. Concentrations of bacteriochlorophyll and total heroes in the culture medium of Chromatium were calculated to be approx. I0 and 0.2 /2M, respectively '-'8. It is most unlikely that both can be synthesized using traces of 02 in the medium. In 1968 , Mori and Sano 6, using a cell-free extract of Chromalium, showed that the conversion of coproporphyrinogen to protoporphyrin was demonstrated under

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aerobic conditions, but not under anaerobic conditions. Some alternative electron acceptors did not replace molecular 02. Maximal protoporphyrin formation occurred at approx. 4 ° °/o 02, indicating a similar K,, for 02 of both Chromatium and mammalian liver mitochondrial enzyme 2. In contrast, Ehteshamuddin 29 reported that the enzyme from a species of aerobically grown Pseudomonas was capable of forming protoporphyrin under anaerobic conditions in the presence of glutathione. Tait 7 showed in 1969 that an extract from Rhodopseudomonas spheroides, grown semi-anaerobically in the light, converted coproporphyrinogen I I I to protoporphyrin I X when incubated anaerobically in the dark with Mg ~+, A T P and L-methionine. Extracts from R. spheroides grown under air in the dark were inactive when assayed under these conditions. Extracts from organisms grown in the light or in the dark formed protoporphyrin in the absence of ATP and methionine when incubated aerobically ~. He suggested further that NAD(P)+ might be the final electron acceptor 3°. These findings are most important and interesting, because the sulfur-containing compound m a y play an important role in anaerobic protoporphyrin formation. However, our experiments using cell-free extracts of Chromatium or aerobically grown Pseudornonas fluoresceus and Pseudomonas arvilla, showed that the conversion of coproporphyrinogen to protoporphyrin was easily demonstrated under aerobic conditions but not under anaerobic conditions, even when glutathione or ATP, Mg 2+ and methionine had been supplemented. The results obtained by Jacobs et al. ~ are in good agreement with our observations with Chrornatium, despite conflicting reports elsewhere. This lack of success could be due either to low activity or instability of the enzyme system involved in protoporphyrin biosynthesis or to difference of bacterial strains, or to the fact that external electron acceptors added in this experiment differed from natural electron acceptors. Tait 3° observed that high concentrations of Tris and borate inhibited the anaerobic formation of protoporphyrin. This could explain our failure to detect anaerobic activity when coproporphyrinogen prepared by NaBH4 was used. Coproporphyrinogenase was partially purified from Chromatium, and some of its properties were examined. The p H optimum was 6.4, more acidic in comparison with the value of p H 7.4-7.7 for mammalian liver enzymes1, is. No evidence was found for the involvement of cofactors in this enzymic reaction. Hg 2+ and Ag + strongly inhibited the activity but the inhibiting mechanism was not known. In spite of property differences, the enzyme found in Chromatium was very similar to that of mammalian liver mitochondria. The role of the 02-requiring enzyme system in protoporphyrin synthesis in Chromatium is now under investigation. We would like to point out the following additional results obtained with another anaerobic bacteria, Desulfovibrio vulgaris. Porphyrin formation from b-aminolevulinic acid was stopped completely at the uroporphyrinogen stage and further conversion not observed. No activity in uroporphyrinogen decarboxylase and eoproporphyrinogenase was detected. The explanation of this dilemma is still very obscure. ACKNOWLEDGEMENTS The authors thank Miss M. Ohara for assistance with the manuscript. This investigation was supported in part by Research Grants from National Institutes of Health GM 11793-02 and 03, Fujiware Foundation of Kyoto University, Waksman Foundation and the Scientific Research Fund of the Ministry of Education of Japan.

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