Protoporphyrinogen oxidation in Rhodopseudomonas spheroides, a step in heme and bacteriochlorophyll synthesis

ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS Vol. 211, No. 1, October 1, pp. 305-311, 1981

Protoporphyrinogen Oxidation in Rhodopseudomonas spheroides, a Step in Heme and Bacteriochlorophyll Synthesis N. J. JACOBS Department

of Microbiology,

Dartmouth

AND Medical

Received

March

J. M. JACOBS School,

Hanover,

New

Hampshire

O$%Vi

5, 1981

Protoporphyrinogen oxidizing activity in photosynthetically grown, bacteriochlorophyll-rich extracts of Rhodopseudomonas spheroides was heat labile, destroyed by trypsin digestion, associated with membranes, and proportional to extract concentration. Substrate specificity was indicated by the inability to oxidize other porphyrinogens. These properties are consistent with an enzymatic reaction. Activity was markedly inhibited by respiratory inhibitors such as cyanide, azide, and hydroxylamine, by reducing agents such as glutathione and 2-mercaptoethanol, and by respiratory substrates such as succinate and NADH. These agents as well as protoporphyrinogen also caused reduction of cytochromes during the assay. Extraction of quinones from membranes with pentane also inhibited protoporphyrinogen oxidation. These results clearly indicate that this oxidation in R. spheroides is closely linked with components of the respiratory electron transport chain, suggesting possibilities for regulation by light and oxygen. Cyanide did not cause inhibition of protoporphyrinogen oxidation by rat liver mitochondria, suggesting different mechanisms for the bacterial and mitochondrial systems. In addition, an enzyme able to link protoporphyrinogen oxidation directly to oxygen was solubilized with Triton from rat liver mitochondria, but not from R. spheroides membranes. Membranes from R. spheroides grown aerobically, when bacteriochlorophyll synthesis does not occur, exhibited about half the protoporphyrinogen oxidizing activity as membranes from photosynthetically grown cells. This activity was also markedly inhibited by cyanide and could not be solubilized with Triton, but was not inhibited by glutathione.

The oxidation of protoporphyrinogen to protoporphyrin is a step in tetrapyrrole synthesis which may have regulatory implications for both the heme and chlorophyll synthesis pathway (Fig. 1). The removal of six hydrogens from the porphyrinogen ring can occur nonenzymatically, but is enzymatic in liver and yeast mitochondria (l-6). An enzyme has been solubilized from mitochondria which uses oxygen as sole hydrogen acceptor and no cofactors were demonstrable (3,4). We found that in Escherichia coli, this oxidation can occur without oxygen, utilizing an anaerobic electron acceptor such as fumarate (7, 8), and that electron transport carriers such as quinones can be involved (9-11). In chlorophyll-containing tissue, proto-

porphyrinogen oxidation has not been studied, although we recently demonstrated the presence of activity in a photosynthetic bacterium (12). In this study, we characterize this reaction in Rhodopseudomonas spheroides grown under photosynthetic, anaerobic conditions where bacteriochlorophyll is synthesized in loo-fold excess over heme (13) and where oxygen cannot serve as oxidant. We also compare this activity to that found in cells grown aerobically where bacteriochlorophyll is not made. Our results suggests that R. spheroides oxidizes protoporphyrinogen by a different enzymatic mechanism than found in mitochondria.

i Supported PCM 79-19520.

The carotenoid-deficient mutant of R. spheroides, strain R26, utilized to minimize interfering pigments,

by National

Science

Foundation

METHODS

Grant

305

306

FIG. 1. Pathway

JACOBS

of heme

and chlorophyll

AND

synthesis.

was generously supplied by R. K. and B. J. Clayton and grown under photosynthetic, anaerobic conditions as previously described (12) for 3 days, when bacteriochlorophyll synthesis was well developed. Aerobic growth was in the same medium, but for 40 h on a shaker as described, when the culture was in the late log phase of growth (12). Extracts were prepared as previously described (12) but with disruption by two passages through a French pressure cell at 4°C. Extracts were sedimented at 12,OOOg for 10 min to remove whole cells and debris and the supernatant solution was designated as crude extract. A membrane fraction was prepared by centrifugation (4°C) at 144,OOOg for 4 h, and resuspending with 50 mM Tris-HCl, pH 7.6, to the original volume. Liver mitoehondria were prepared as previously described from male Sprague-Dawley rats (14). Enzyme preparations were stored at -70°C before use. The assay for protoporphyrinogen oxidation, described previously (8), involves adding the colorless porphyrinogen to extract at room temperature. Cuvettes were placed at 3’7°C and scanned at intervals for appearance of the four-banded spectrum of oxidized porphyrin, using as reference a cuvette containing the entire reaction mixture, minus porphyrinogen. In the present study only the absorption peak at 633 nm was used to calculate the amount of porphyrin formed, since other pigments interfered with measurements at the other peaks (8). The peak at 540 nm overlapped with the cytochrome c peak under some conditions, but was also monitored to insure agreement with the changes at 633 nm. This difference spectrum technique also allowed determination of cytochrome reduction from the reduced minus oxidized spectrum. Unless indicated otherwise, the rates of enzymatic protoporphyrinogen oxidation were corrected for the nonenzymatic rate by subtracting the rate of oxidation by heat-inactivated extract which was prepared by heating to 85’C for 30 min, with stirring, and then sonication (12) for a few seconds to disperse coagulated protein. Assays were conducted in open 3-ml cuvettes without stirring during incubation. Mesoporphyrin IX and uroporphyrin I (97% pure) (Porphyrin Products, Logan, Utah) were reduced and their extinction values and rates of oxidation were determined as described for protoporphyrin (8). Digestion with trypsin was accomplished by adding 5 mg crude trypsin

JACOBS (Sigma Chemical Co., St. Louis, MO.) to 48 mg protein of particles from photosynthetically grown cells in 8.3 ml of 50 mM Tris-HCl (pH 7.6) and incubation under an atmosphere of prepurified nitrogen gas for 4 h at 37°C. Quinones were extracted at room temperature from membranes of photosynthetically grown cells with 35 ml of pentane (containing 0.5% methanol) per 40 mg (dry weight) of lyophilized membranes using a tissue homogenizer (15). Membranes were centrifuged, the solvent removed, and the extraction repeated twice. Analysis of the solvent for quinone content (16) revealed most of the quinone was removed in the first extraction. The extracted particles were dried in vucuo (15) and resuspended in 50 mM Tris-HCl, pH 7.6. Reconstitution was attempted by homogenizing 300 mg of extracted dried particles in 25 ml of pentane containing 12.5 mg coenzyme QlO (15), followed by drying and resuspension in Tris buffer as above. &enzyme QlO (bovine heart) was from Sigma. Respiration was measured with an oxygen electrode (12).

RESULTS

Protoporphyrinogen Oxidation Extracts of Photosynthetically Cells

by Grown

The rate of protoporphyrin formation from protoporphyrinogen in membranes prepared from photosynthetic cells was markedly decreased if the membranes were inactivated by heat (Fig. 2). Digestion of the particles with trypsin decreased the rate of protoporphyrinogen oxidation to that exhibited by the heated control. The rate of enzymatic (heat labile) protoporphyrinogen oxidation was proportional to the amount of extract between 2 and 6 mg extract protein per milliliter of assay mixture. Lower amounts gave little activity above the heated control, and larger amounts could not be measured due to interference from other pigments. The activity was located in the membrane, rather than the supernatent fraction (Fig. 3). Activity was proportional to amount of membrane added, and the specific activity was 3.0 nmol protoporphyrin produced per hour per milligram of membrane protein (data not shown). When the assay temperature was de-

Rhodopseudomonas

spheroides

PROTOPORPHYRINOGEN

20

FIG. 2. Oxidation of protoporphyrinogen by membrane fraction of photosynthetically grown cells. The reaction mixture, in open cuvettes, contained: 56 mM Tris-HCl buffer, pH 7.6; 1 mM EDTA; approximately 130 nmol of protoporphyrinogen; and 12.2 mg protein of particles either unheated (0) or heated (X), and water to a total volume of 3.0 ml. Nanomoles of protoporphyrin formed in the cuvette are shown.

creased to lO”C, the enzymatic rate was 35% of that observed at 37°C. Dialysis at 4°C of extracts for 48 h against two changes of 50 mM Tris-HCl buffer, pH 7.6, did not substantially decrease enzymatic activity. When mesoporphyrinogen IX or uroporphyrinogen I was substituted as 130 120 110

307

OXIDATION

60

100

160

0

Time

(min)

40

80

FIG. 4. Effect of glutathione and cyanide on protoporphyrinogen oxidation. Reaction mixture as in Fig. 2, with 10 mg protein of R. spheroides extract or 3.5 mg protein of mitochondria either heated (0) or unheated (0) as shown. Where indicated (A) 5 mM glutathione or (X) 8 mM KCN was added to the unheated extract. Glutathione (1 mM) was present in all the mitochondrial samples to minimize autooxidation. Equivalent inhibition of the R. spheraides system was observed at 0.1 mM KCN.

substrate, these unphysiological porphyrinogens were oxidized very slowly, and as rapidly by heated as by unheated extracts, indicating autooxidation rather than enzymatic oxidation. This suggests a specificity for protoporphyrinogen as substrate.

Effect of Reducing Agents and Respiratory Substrates in Extracts Photosynthetic Cells

20

60

100

140

200

260

Time lminl

FIG. 3. Particulate nature of protoporphyrinogen oxidizing activity. Assay mixture as in Fig. 2, with 13 mg protein of particulate fraction (solid line) or 7 mg of supernatent fraction (dashed line) either heated (X) or unheated (O), where indicated. Fractions were prepared by centrifugation of 14 ml of crude extract for 4 h at 144,600g at 4°C and resuspending the pellet in 14 ml of 50 mM Tris-HCl buffer, pH 7.6. One milliliter of the clear supernatent or pigmented particulate fraction was added to the assay mixture.

120

of

Addition of glutathione to reaction mixtures markedly inhibited protoporphyriogen oxidation in photosynthetic membranes (Fig. 4). Other reducing agents, such as 2-mercaptoethanol and dithiothreitol (5 mM) were equally inhibitory. These reducing agents do not cause significant inhibition of the mammalian enzyme (3, 4), or the E. co2i system (12, 17) which are assayed in the presence of reducing agents to minimize autooxidation. We also found that the addition of respiratory substrates such as NADH (2 InM) or succinate (10 mM) to the assay mixture also caused the rate of protoporphyrinogen oxidation to decrease by more than 80%.

JACOBS

AND

JACOBS

it was not possible to determine the effect of protoporphyrinogen on cytochrome c reduction by this method (Fig. 6). Effect of Respiratory Protoporphyrinogen

Wavelength (nm)

FIG. 5. Reduction of eytochromes in R. spheroides by glutathione, NADH, and hydrosulfite. Reaction mixture as in Fig. 2 with crude extract (9.9 mg protein) of photosynthetic cells but no protoporphyrinogen added. Glutathione (5 mM) or NADH (2 mM) were added to the sample cuvette, and the difference spectra were recorded after 20 min at room temperature. Complete cytochrome reduction was achieved by addition of a few crystals of sodium hydrosulfite. Addition of 5 mM 2-mercaptoethanol caused identical cytochrome reduction as shown for glutathione.

Reduction Assay

of Cytochromes

during

Inhibitors on Oxidation

Cyanide also caused marked inhibition of protoporphyrinogen oxidation by extracts of photosynthetically grown R. spheroides. Cyanide concentrations as low as 0.1 mM were as inhibitory as 8 IIIM (Fig. 4). When the cyanide concentration was decreased to 0.05 mM, inhibition decreased from 100 to 85%. Cyanide was equally inhibitory if added before or after addition of substrate or if tested on dialyzed membrane preparations. Azide and hydroxylamine (tested at 10 mM) were as inhibitory as cyanide. Cyanide was not significantly inhibitory to protoporphyrinogen auto oxidation, tested in heated extracts, or to the slow rate of auto oxidation of mesopor-

the

Since the inhibitory effect of reducing agents and respiratory substrates might be related to their effect on electron transport components, we studied the effect of these agents on cytochromes in photosynthetic membranes. Glutathione and NADH caused partial reduction of cytochromes c and b with cy peaks at 552 and 560 nm when added to the assay mixture in the absence of protoporphyrinogen (Fig. 5). Addition of protoporphyrinogen solution or succinate also caused partial cytochrome reduction (Fig. 6). Demonstration of cytochrome b reduction by protoporphyrinogen required addition of cyanide to prevent reoxidation of cytochromes. Since the addition of cyanide without added substrate caused cytochrome c reduction under these conditions,

500 -

600 Wavelength

(nm)

FIG. 6. Reduction of cytochrome by protoporphyrinogen and succinate in membranes of photosynthetically grown cells. Contents of sample and reference cuvette as in Fig. 2, with 9.5 mg of membrane protein which had been dialyzed (40 h at 4°C against 50 mM Tris-HCl, pH 7.6) to remove endogenous substrates. Cyanide was added to prevent reoxidation of reduced cytochromes. Solid line: KCN (0.5 mM) added to sample cuvette 40 min prior to recording spectra. Cyanide addition causes reduction only of cytochrome c (550 nm) under these conditions. Dashed line: KCN (0.5 mM) added to sample cuvette 40 min prior to recording, and protoporphyrinogen (53 nmol) added 18 min prior to recording. Cytochrome b (560 nm) becomes partially reduced on addition of protoporphyrinogen. An identical pattern of cytochrome reduction was observed if 10 nmol succinate was substituted for protoporphyrinogen.

Rhodopseudommm

sphe-roides

PROTOPORPHYRINOGEN

phyrinogen in extracts. Cyanide did not destroy protoporphyrinogen, since exposure of the cuvette containing cyanide (Fig. 4) to bright light following the 2-h incubation period caused a rapid oxidation to the level of protoporphyrin found in the cuvette without cyanide. One explanation for the mechanism of cyanide inhibition is that protoporphyrinogen oxidation requires participation of a cyanide-sensitive component of the respiratory chain We therefore tested the effect of cyanide on respiratory activity. Respiration with 3.7 mM succinate as substrate (1.4 nmol Oz consumed/min/mg protein) was 90% inhibited by cyanide (tested at 4 mM). As indicated above, cyanide also caused partial cytochrome reduction when added to membranes (Fig. 6). In mitochondria, cyanide did not cause significant inhibition of protoporphyrinogen oxidation (Fig. 4). The enzyme solubilized from mitochondria was not inhibited by cyanide (4). We found that other inhibitors, such as the metal chelators o-phenanthroline (0.5 mM), thenoyl trifluoroacetone (2 mM), or EDTA (5 mM), and the respiratory inhibitors antimycin A (0.2 mM) or 2-heptyl-4hydroxy quinoline-N-oxide (0.4 mM), did not cause appreciable inhibition of protoporphyrinogen oxidation in R. spheroides particles. These respiratory inhibitors are not as effective as cyanide as inhibitors of respiration in R. spheroides

(x3-20). Effect

of Quinone

Extraction

To determine a possible role of quinones, as previously found for this reaction in E. coli (11, 12), we extracted quinones from photosynthetic particles with pentane. This caused a considerable decrease in the rate of enzymatic protoporphyrinogen oxidation. In two separate extractions on two batches of particles, activity was decreased by 84 and 74% by pentane extraction. Analysis of the pentane extract showed that quinone had been extracted by this procedure. In addition, extracted particles no longer exhibited ability to

309

OXIDATION

cause reduction of cytochromes b and c by succinate (measured as in Fig. 5), which was readily demonstrable in unextracted particles. However, preliminary attempts to reconstitute protoporphyrinogen oxidation by addition of authentic coenzyme QlO (15) were not successful. Therefore, a role for quinones has not been confirmed. We have not yet determined if other lipid-soluble factors are involved. Attempts

to Solubilixe

the Activity

Equivalent amounts of membrane protein from photosynthetically grown R. spheroides and rat liver mitochondria were extracted in parallel, using a combination of Triton X-100 and sonication by the method previously described for solubilizing the protoporphyrinogen oxidizing enzyme from liver mitochondria (4). Following these extractions, the 160,000~ supernatent fraction of the Triton extract of mitochondria exhibited enzymatic activity at a level of approximately 10 nmol protoporphyrin formed per hour per milligram mitochondrial protein when assayed in the presence of 5 mM glutathione. This indicated solubilization of the mitochondrial enzyme. In contrast, no enzymatic activity was demonstrable in the 160,OOOg supernatent fraction of the Triton extract of R. spheroides membranes, when assayed either in the presence or absence of glutathione, indicating that no enzyme with similar properties to the mitochondrial enzyme was solubilized from R. spheroides membranes. In addition, no enzymatic activity was detected in the particulate residue, indicating that membrane disruption destroyed activity. Protoporphyrinogen Oxidation Aerobically Grown Cells

in

We have conducted some studies on cells of this bacterium grown aerobically, when bacteriochlorophyll is not synthesized. Membranes prepared from such cells were capable of enzymatic protoporphyrinogen oxidation when tested in the aerobic assay exhibiting a rate of 1.6 nmol protoporphyrin produced per hour per milligram

310

JACOBS

AND

protein which was proportional to membrane concentration. This oxidation was more than 90% inhibited by cyanide (tested at 0.5 mM). Extraction of aerobic membranes with Triton X-100, as described above, yielded no evidence of an enzyme such as found in mitochondria. In contrast to photosynthetic membranes, aerobic membranes exhibited only slight (less than 20%) inhibition by 5 mM glutathione. This lack of glutathione inhibition in the aerobic membranes, when compared to its strong inhibition in the photosynthetic membranes, was readily understood when we observed that glutathione, which causes considerable cytochrome reduction in photosynthetic membranes (Fig. 5), did not cause cytochrome reduction when added to aerobic membranes. DISCUSSION

Protoporphyrinogen oxidation by extracts of this bacterium exhibits several characteristics suggestive of enzyme involvement. For instance, it is heat labile, destroyed by trypsin digestion, specific for protoporphyrinogen as substrate, present only in the membrane fraction, and proportional to membrane concentration. The inhibitory effects of respiratory inhibitors, reducing agents, respiratory substrates, and quinone extraction and the effects of Triton extraction suggest a mechanism for this oxidation and indicate how the R. spheroides system differs from the mitochondrial enzyme. Low concentrations of cyanide inhibit protoporphyrinogen oxidation in R. spheroides. Azide and hydroxylamine also inhibit. These compounds are known inhibitors of the cytochrome c oxidase system in R. spheroides (M-20) and we have confirmed this by demonstrating marked inhibition of succinate oxidation and reduction of a portion of the cytochromes upon addition of cyanide to R. spheroides membranes. Cyanide is not acting as a metal chelator, since other metal chelators are not inhibitory. It is significant that when the cyanide concentration was decreased from 0.1 to 0.05 mM, inhibition of protoporphyrinogen oxidation decreased from

JACOBS

100 to 85%. The succinoxidase system of is 80% inhibited by 0.05 mM cyanide (19). These observations are consistent with the suggestion that protoporphyrinogen oxidation in R. spheroides requires participation of the electron transport chain, with the cyanide-sensitive cytochrome oxidase ultimately linking to oxygen. Two further observations compatible with this suggestion are the inhibition of protoporphyrinogen oxidation upon extraction of quinones, and the ability of protoporphyrinogen to cause reduction of cytochromes during the assay. The effects of reducing agents such as glutathione also support this suggestion, since they can best be explained in terms of an interaction between protoporphyrinogen oxidation and components of the respiratory chain. In photosynthetic membranes, glutathione markedly inhibits protoporphyrinogen oxidation and causes reduction of the cytochrome components. This represents further evidence that protoporphyrinogen cannot be oxidized if the electron transport carriers are in the reduced form. This interpretation of the glutathione effect was confirmed when we observed that in aerobic membranes, glutathione caused only slight inhibition of protoporphyrinogen oxidation, and did not cause reduction of cytochrome pigments. These findings explain our previously published observation (12) that protoporphyrinogen oxidation could not be demonstrated in photosynthetic membranes of R. spheroides when the assay was conducted in the presence of glutathione, but that activity in aerobic membranes was demonstrable. The inhibitory effect of respiratory substrates such as NADH and succinate can also be explained in terms of their demonstrated ability to cause reduction of the components of the respiratory electron transport chain, thereby preventing protoporphyrinogen oxidation. This observation could have implications for the regulation of bacteriochlorophyll and heme synthesis. Physiological states which cause accumulation of respiratory substrates could cause inhibition of tetrapyrolle biosynthesis through such a mechanism. Our R. spheroides

Rhodapseudamonas

spheroides PROTOPORPHYRINOGEN

findings could also have implications for the regulation of tetrapyrolle synthesis by light and oxygen (13, 21-25), since both these factors are known to alter the redox state of electron transport carriers. With regard to the mechanism of the reaction, our data clearly indicate a linkage between protoporphyrinogen oxidation and the electron transport chain in R. spheroides. An important consequence of this linkage is that protoporphyrinogen oxidation is markedly inhibited if the electron transport carriers are in the reduced form. However, the exact mechanism of this linkage must await further study. Our findings are consistent with protoporphyrinogen entering the electron transport chain somewhere in the region of quinones, nonheme iron, and cytochromes b and c, and being oxidized in a manner similar to a respiratory substrate such as succinate, but at a much slower rate. There may be a specific enzyme for linking protoporphyrinogen to the electron transport chain, which would explain the specificity for protoporphyrinogen. However, there is no enzyme such as found in mitochondria which links directly to oxygen and can be solubilized with Triton. Lack of cyanide inhibition also distinguishes the mitochondrial from the R. spheroides system. When R. spheroides was grown aerobically, when bacteriochlorophyll synthesis does not occur, membranes from aerobic cells were capable of oxidizing protoporphyrinogen in the aerobic assay at a rate which was slightly more than half that exhibited by membranes from photosynthetic cells. Obviously, the slight difference in specific activity as measured in this assay does not explain the loo-fold difference in chlorophyll synthesis between aerobic and photosynthetically grown cells. The mechanism of oxidation in both membranes is apparently similar, since both were inhibited by cyanide. ACKNOWLEDGMENTS We are grateful Clayton of Cornell supplying mutant

to Drs. R. K. Clayton and B. J. University, Ithaca, New York, for R26 and advice on its cultivation.

311

OXIDATION REFERENCES

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