Heme biosynthesis is coupled to electron transport chains for energy generation

Abstract

Cellular energy generation uses membrane-localized electron transfer chains for ATP synthesis. Formed ATP in turn is consumed for the biosynthesis of cellular building blocks. In contrast, heme cofactor biosynthesis was found driving ATP generation via electron transport after initial ATP consumption. The FMN enzyme protoporphyrinogen IX oxidase (HemG) of Escherichia coli abstracts six electrons from its substrate and transfers them via ubiquinone, cytochrome bo₃ (Cyo) and cytochrome bd (Cyd) oxidase to oxygen. Under anaerobic conditions electrons are transferred via menaquinone, fumarate (Frd) and nitrate reductase (Nar). Cyo, Cyd and Nar contribute to the proton motive force that drives ATP formation. Four electron transport chains from HemG via diverse quinones to Cyo, Cyd, Nar, and Frd were reconstituted in vitro from purified components. Characterization of E. coli mutants deficient in nar, frd, cyo, cyd provided in vivo evidence for a detailed model of heme biosynthesis coupled energy generation.

Heme is an essential cofactor of enzymes in electron transport chain mediated energy generation. It is synthesized using a highly conserved pathway (1). The penultimate step of heme biosynthesis—the conversion of protoporphyrinogen IX (proto’gen) via the abstraction of six electrons into protoporphyrin IX (proto)—is catalyzed by protoporphyrinogen IX oxidases (PPO; EC 1.1.3.4). An O₂-dependent PPO, usually encoded by the hemY gene, is missing in Escherichia coli (2). Over 30 yr ago an oxygen-independent PPO activity was detected in cell-free extracts of E. coli which was dependent on electron transfer to nitrate or fumarate (3–5). The observation indicated the coupling of anabolic heme biosynthesis to catabolic electron chain-driven ATP synthesis. Complementation of the proto accumulating E. coli strain SASX38 yielded the hemG gene (6). Because of the small size of the deduced HemG protein (M_r = 21,200) and the fact that no PPO activity has been shown for the isolated peptide, it was assumed that HemG may be a subunit of a larger PPO complex (2). Additionally, HemG does not share any amino acid sequence homology to oxygen-dependent HemY. Very recently it was reported that purified recombinant E. coli HemG carries menadione-dependent protoporphyrinogen IX oxidase activity (7). The recombinant protein was described as completely water soluble. A structural model based on the homology to long chain flavodoxins was proposed (7). Respiratory reactions possibly coupled to the HemG reaction are usually used to drive membrane-bound electron transport chain for the formation of an electrochemical ion gradient. This membrane-localized gradient, called proton motive force, provides the energy for ATPase to generate ATP from ADP and P_i (8, 9). Electron transport chains consist of numerous primary dehydrogenases and quinone-linked terminal oxidoreductases. Primary dehydrogenases in E. coli include those for NADH, lactate, glucose, formate, and hydrogen (9). The three quinones of E. coli are ubiquinone (UQ), menaquinone (MQ), and demethylmenaquinone (DMQ) (9). At high O₂ tensions in the growth medium, cytochrome bo₃ (EC 1.10.3; Cyo) dominates over all other terminal oxidases. At low O₂ tensions the second cytochrome oxidase, termed bd (EC 1.10.3; Cyd), is present in the cytoplasmic membrane (10–15). Both enzyme systems are known to couple the four-electron reduction of O₂ to two H₂O molecules with the formation of a proton potential via the membrane (13, 14). When O₂ is absent, E. coli is capable of utilizing nitrate, nitrite, TMAO (trimethylamine N-oxide), DMSO (dimethylsulfoxide), and fumarate as alternative terminal electron acceptors (9). The dissimilatory nitrate reductase (EC 1.6.6.1) NarGHI is a membrane-bound enzyme complex consisting of the nitrate-reducing subunit NarG, the electron-transfer subunit NarH, and the quinol-oxidizing subunit NarI (15–18). Fumarate reductase (EC 1.3.1.6; Frd) of E. coli is a membrane-bound flavoprotein catalyzing the cytoplasmic reduction of fumarate to succinate, as well as the oxidation of quinols in the membrane (9, 19). The biochemical principles of coupling protoporphyrinogen IX oxidation in E. coli to the outlined electron transport chains were completely unknown.

Results and Discussion

E. coli HemG Is a Membrane Associated Protoporphyrinogen IX Oxidase.

In order to purify E. coli PPO we first established an enzyme assay using PPO active cell-free extract. However, this assay always required the presence of fumarate reductase and menaquinone for PPO activity. Hence, we replaced those with 2.5 μM of the artificial electron acceptor triphenyltetrazolium chloride (TTC, Fig. 1). In this context several of the tested potential electron acceptors [i.e. 2, 6-dichloroindophenol (DCIP), phenazinmethosulfate (PMS), menadione, and vitamin K1, respectively] were found to directly oxidize the substrate in the absence of HemG and were therefore not used in the activity assay system. TTC was the only stable electron acceptor utilized by HemG and consequently employed in this study. Corresponding chemical structures are given in Fig. 1. Using the TTC-based enzyme test system, PPO activity of 247 pmol proto/mg protein/h was found associated with an isolated membrane fraction of E. coli (Table S1). Crude cell-free extract (53 pmol proto/mg protein/h) and the cytosolic fraction (11 pmol proto/mg protein/h) were significantly less active. Membrane fraction proteins were solubilized using the detergent Thesit® (2-dodecoxyethanol) and further purified via anion exchange chromatography. Obtained fractions harboring the highest specific PPO activity were analyzed for the identity of contained proteins by mass spectrometry. The most frequently detected peptides in the analyzed samples belonged to the HemG protein (Table S2). Nevertheless, the question still remained whether the strong PPO activity of the purified membrane fraction is caused by HemG alone or whether the membrane fraction contained other components contributing to the 6 e^- oxidation of proto’gen. The B. megaterium genome does neither possess a hemG gene nor the enzymes of anaerobic respiration including fumarate and nitrate reductase. Bacilli are synthesizing proto using the oxygen-dependent flavin-enzyme HemY (20). In order to exclude association of E. coli HemG with additional potential PPO subunits from E. coli the HemG protein was produced fused to a His-tag in B. megaterium (Fig. 2). With TTC as electron acceptor B. megaterium cell-free extract containing recombinant E. coli, HemG revealed the formation of 1.2 nmol proto/mg protein/h. Purification via affinity and gel permeation chromatography yielded apparently pure protein (Fig. 2A). As expected, purified HemG solely revealed enzyme activity in the presence of TTC as electron acceptor (23 nmol proto/mg protein/h). Removal of the fused His-tag via protease digestion did not change the catalytic properties, neither with TTC (21 nmol proto/mg protein/h) nor with purified fumarate reductase as electron acceptors (55 nmol proto/mg protein/h). Consequently, all further experiments were conducted with His-tagged HemG. The Michaelis-Menten constant K_M as well as the maximal velocity V_max were determined with menaquinone as electron acceptor. The K_M value of 17.3 μM for the substrate was obtained. However, the K_M of the substrate was 15 times higher compared to tobacco oxygen-dependent HemY PPO (K_M of 1.17 μM) (21). On the other hand, the calculated maximal velocity V_max of HemG of 960 μM h^-1 mg^-1 was 4-fold higher than that of tobacco HemY with 256.2 μM h^-1 mg^-1 (21). Acifluorfen is a herbicide inhibitor of oxygen-dependent PPOs of the HemY-type (22). Applied in concentrations of up to 250 μM, the compound did not influence HemG activity with TTC (19 nmol proto/mg protein/h) and fumarate reductase (52 nmol proto/mg protein/h) as electron acceptor. Fumarate reductase subunit FrdA was reproducibly detected in PPO-activity containing fractions of the HemG purification from the membrane fraction of E. coli and from recombinant E. coli. Corresponding peptides are given in Table S2. These observations indicated protein-protein interactions between HemG and E. coli FrdA. Gel permeation chromatography of purified E. coli HemG in the presence of 2% Thesit® on Superdex 200 HR 10/30 revealed an identical elution point as the marker proteins yeast alcohol dehydrogenase (M_r = 150,000) indicating a M_r of ∼150,000 and a hexameric oligomerization state. A UV-Vis spectral analysis of the purified protein showed in agreement with a recent investigation of recombinant HemG (7) a typical spectrum for a flavin cofactor (Fig. 3A). The flavin was identified as noncovalently bound FMN using HPLC analyses (Fig. 3B).

Fig. 1.

Electron acceptors tested for E. coli HemG. The structures of the following electron acceptors used in this work are depicted: 2, 6-dichloroindolphenol (DCIP) with an E^0′ of +237 mV, menadione (provitamin K₃, E^0′ = -205 mV), phenazinmethosulfate (PMS, E^0′ = +80 mV), 2, 6-triphenoltetrazoliumchloride (TTC, E^0′ = -80 mV), phylloquinone (vitamin K₁, E^0′ = -170 mV), menaquinone (vitamin K₂, E^0′ = -74 mV), and ubiquinone (coenzyme A, E^0′ = +110 mV). HemG cofactor FMN has E^0′ = -190 mV. Redox potentials are given for the acceptor-donor couples of the free compounds (40, 41). Protein environments might individually change the corresponding values.

Fig. 2.

Recombinant production of E. coli HemG in B. megaterium. (A) E. coli HemG after recombinant production in B. megaterium and affinity chromatographic purification was subjected to 15% SDS-PAGE. Lane 1, molecular weight standard; lanes W1–W3, Ni-NTA Superflow resin chromatography fractions. Lanes E1, E2, distinct bands of M_r ∼ 22,000 corresponding to the size of His_6x-tagged HemG was observed. (B) PPO activities of fractions from the purification of HemG by affinity chromatography. PPO activities were obtained as described in Materials and Methods. Proto formation was depicted with TTC as electron acceptor. W1–3: activities corresponding to the fractions as shown in (A). E1–2: activities of recombinant HemG corresponding to the fractions obtained in (A). Arbitrary units: relative fluorescence units with t = 0 min subtracted from t = 60 min.

Fig. 3.

Spectral analysis of E. coli HemG and the bound flavin cofactors. (A) A UV-Vis spectrum of purified HemG (black line) and extracted cofactor (red line) against elution buffer was recorded. The spectra with characteristic peaks at 366 and 433 nm indicate the presence of a flavin cofactor (19). (B) HPLC analysis for the identification of the flavin cofactor of HemG. The retention time for the cofactor FAD (flavine adenine dinucleotide) was 7.25 min (blue) and 10.1 min for FMN (flavine mononucleotide) (red). The purified cofactor from the HemG fraction was detected at 10.1 min and thus identified as FMN (green).

Ubiquinone and Menaquinone Are Electron Acceptors of HemG Catalysis.

The quinol analogue pentachlorophenol (PCP, Fig. 1) inhibits fumarate reductase via blocking the quinone binding site of the enzyme (9). At a concentration of 1 μM it was also a strong inhibitor of HemG activity when tested with fumarate reductase as electron acceptor (Table 1). In contrast, HemG tests with 1 μM TTC as electron acceptor remained unaffected (22 nmol proto/mg protein/h). An inhibition of quinone-dependent electron transfer from HemG to fumarate reductase was concluded. Fumarate reductase employs menaquinone as electron carrier. However, other terminal oxidoreductases including the cytochrome bd complex couple to ubiquinone. Both quinones were successfully tested as electron acceptors for catalysis with purified recombinant HemG (Table 1).

Table 1.

In vitro reconstitution of HemG coupled electron transport chains of E. coli with purified components and PPO activities in E. coli electron transport chain mutants

E. coli HemG + proto’gen * + terminal oxidoreductase †	+ quinone †+ electron acceptor †	Proto formed [nmol/mg protein/h]
n. a. †		n.d.‡
Cytochrome bo3 oxidase	+ UQ +O2	51.1 ± 7.3
n. a. †	+ UQ +O2	28.3 ± 3.3
n. a. †	+O2	n.d.‡
n. a. †	+ UQ	25.5 ± 2.4
Cytochrome bo3 oxidase	+ UQ	25.2 ± 3.3
Cytochrome bo3 oxidase	+O2	40.8 ± 5.4
Cytochrome bd oxidase	+ UQ +O2	86.9 ± 6.6
n. a. †	+ UQ +O2	44.7 ± 4.4
n. a. †	+O2	n.d.‡
n. a. †	+ UQ	41.7± 3.2
Cytochrome bd oxidase	+ UQ	50.7 ± 3.1
Cytochrome bd oxidase	+O2	58.9 ± 4.3
Fumarate reductase	+ MQ + fumarate	52.4 ± 6.6
n. a. †	+ MQ + fumarate	22.4 ± 2.1
n. a. †	+ fumarate	n.d.‡
n. a. †	+ MQ	21.3 ± 2.3
Fumarate reductase	+ MQ	10.1 ± 1.2
Fumarate reductase	+ fumarate	23.1 ± 2.3
Fumarate reductase	+ PCP § + fumarate	n.d.‡
Nitrate reductase	+ MQ + nitrate	61.7± 9.6
n. a. †	+ MQ + nitrate	16.2 ± 2.2
n. a. †	+ nitrate	n.d.‡
n. a. †	+ MQ	15.4 ± 1.6
Nitrate reductase	+ MQ	12.3 ± 1.1
Nitrate reductase	+ nitrate	21.4 ± 2.6
Cell-free extract of E. coli strain and [relevant genotype] ¶		Proto formed [pmol/mg protein/h]
wild-type E. coli	+O2	62.5 ± 7.0
FB20172 [cyoB∷kan]	+O2	31.6 ± 4.2
FB20228 [cydA∷kan]	+O2	54.4 ± 5.5
DS253 [Δcyd∷cam Δcyo∷kan]	+O2	n.d. ‡
G0105 [Δ(cydAB)∷cyo123]	+O2	n.d. ‡
wild-type E. coli	+ fumarate	56.0 ± 4.0
DW35 [ΔfrdABCD]	+ fumarate	27.2 ± 2.0
wild-type E. coli	+ nitrate	51.3± 5.5
JCB4023 [narG::ery ΔnapABnarZ∷Ω]	+ nitrate	33.8 ± 2.0

^*Thirty pmol purified E. coli HemG were incubated with 30 pmol of purified terminal oxidoreductase and 10 μM proto’gen under conditions as outlined in Materials and Methods. O₂ was introduced by vigorous shaking of the reaction vessel. Where indicated 1.8 μM MQ or UQ were added, respectively. Fumarate or nitrate was employed at a concentration of 1 mM, respectively. All experiments were performed with three independent HemG and terminal oxidoreductase preparations in triplicate.

^†As indicated below, n.a. = no further additions.

^‡n.d. = not detectable; the detection limit of the employed test was below 10 pmol/mg protein/h.

^§Pentachlorophenol (PCP) was first titrated from 1 nM to 10 mM for specific inhibition of fumarate reductase without direct reaction with the substrate and further used at specific concentrations of 1 μM.

^¶All employed mutant strains and the wild-type strain were grown in parallel under the required conditions as detailed in Materials and Methods. For most of the E. coli mutant strains the parental strains were analyzed in parallel. Since no obvious differences between these strains and BL21-DE3 concerning their PPO-activity were observed, BL21-DE3 was used as the general wild-type strain. Cell-free extracts were prepared. Hundred μg protein were incubated with 10 μM proto’gen and appropriate electron acceptors 1 mM fumarate, 1 mM nitrate and O₂ under active aeration. All experiments were performed with three independent experiments done in triplicate.

Purified Fumarate and Nitrate Reductase, Cytochrome bd and Cytochrome bo are Electron Acceptors of E. coli HemG.

Next, it was investigated if purified E. coli HemG transfers electrons via the identified quinones towards terminal oxidoreductases of the E. coli respiratory chains and, consecutively, to various terminal electron acceptors. For this purpose, fumarate reductase, nitrate reductase, cytochrome bd oxidase, and cytochrome bo oxidase of E. coli were recombinantly produced and affinity chromatographically purified as described before (16, 19, 23). In combination with the respective quinones and electron acceptors they were analyzed for the ability to accept electrons from the catalytically active E. coli HemG in vitro. Proto formation was monitored spectroscopically. All four terminal oxidoreductases accepted electrons from the HemG catalysis (Table 1). These results clearly demonstrate the coupling of heme biosynthesis to respiratory electron transport and consequently to proton gradient formation. Interestingly, the addition of quinones to the enzyme complexes was not essential in most tested cases. Both purified oxygen-dependent cytochrome oxidases sustained up to 67% (Cyd) and 78% (Cyo) of HemG activity in the absence of ubiquinone, indicating a tight association of the quinones with the enzyme complexes during preparation. This tight quinone binding by both cytochrome oxidases was described several times before (23, 24). Similar observations were made for respiratory nitrate reductase (17). In control reactions the end electron acceptors (nitrate, fumarate, and oxygen) did not directly accept electrons from HemG catalysis (Table 1).

Electron Transfer from E. coli HemG in Vivo.

To confirm the results of our in vitro experiments, additional in vivo experiments using appropriate E. coli mutant strains were performed. Cell lysates of E. coli mutants deficient in the terminal oxidoreductases were investigated for their PPO activity (Table 1). First, the strains were grown under conditions allowing for efficient cell growth of the mutants. Afterwards, the cultures were shifted to growth conditions which allow for a strong expression of the phenotype of interest.

The results of those in vivo experiments confirmed the results of the in vitro experiments (Table 1). All mutants deficient in terminal oxidases were found reduced in PPO activity when tested with the corresponding terminal electron acceptor. However, only in the case of the cyo/cyd double mutant PPO activity was completely abolished. Mutant variants deficient in either Cyo or Cyd retained 50 and 87% of wild-type activity. It was concluded that the PPO electron flow towards oxygen remains largely intact as long as one out of the two cytochrome oxidases is present in the cell. Partial cross complementation of cyo and cyd mutants was observed before (25, 26). The clear cut HemG deficiency in the cyo/cyd double mutant revealed exclusive coupling of HemG to these two systems under aerobic growth conditions. The mutants deficient in fumarate reductase or all three nitrate reductases (narGHI, narXYZ, napFDAGHCB) were capable of sustaining 48% of PPO activity and 65% of wild-type activity, respectively. It is possible that the remaining terminal oxidoreductases in the mutant strains take up electrons via their quinones and transfer them to residual amounts of terminal electron acceptors. Fumarate is a product of the TCA (tricarboxylic acid) cycle and consequently synthesized in the nitrate reductase mutant. Moreover, E. coli possesses additional anaerobic terminal oxidoreductases including TMAO- (TorA), DMSO- (DmsABC) and nitrite- (NrfABCDEFG) reductases (9, 15). In conclusion, the genetic analysis of diverse E. coli respiratory-chain mutant strains clearly demonstrated the dependence of HemG catalysis and thus of the overall heme biosynthesis on respiratory electron transport.

A Model for Electron Transport Coupled Heme Biosynthesis.

The complete in vitro reconstitution of potential electron transport chains for aerobic and anaerobic HemG activity in combination with genetic investigations led to a model for respiratory chain driven PPO activity in E. coli (Fig. 4). Interestingly, the system couples the biosynthesis of the cofactor heme to respiratory systems which in turn are highly dependent on hemes. Clearly, ATP formation is connected with this biosynthetic step due to the proton translocation catalyzed by most of the terminal oxidoreductases (8–16). Heme biosynthesis in E. coli, starting from glutamyl-tRNA, requires eight ATP molecules per formed protoporphyrinogen IX during the initial charging of tRNA^Glu with glutamate (1). Consequently, the observed coupling of HemG to oxidative phosphorylation might balance this ATP consumption. Clearly, heme biosynthesis is not a dominating cellular process in E. coli and consequently ATP generation via HemG coupled electron transfer might not contribute quantitatively to its energy charge. Nevertheless, a principle of an anabolic, biosynthetic pathway driven catabolic proton gradient formation and consequently ATP generation was biochemically unraveled. Because of the employment of oxygen, nitrate, and fumarate as electron acceptors, heme biosynthesis is ensured in aerobic as well as anaerobic environments. Our model is further substantiated by the observed protein-protein interactions of immobilized HemG with fumarate reductase subunit FrdA. Such protein-protein interactions might provide the basis for the assembly of the electron transport chain machinery. Consequently, heme biosynthesis with HemG might be part of the recently described respirazones in E. coli, mobile patches of the plasma membrane where respiratory enzymes like cytchrome bd are concentrated (27). Moreover, the necessary quinones were found associated with the terminal oxidoreductase. In agreement, the addition of the quinol analogue, PCP, abolished PPO activity. PCP is also known to inhibit nitrate reductase and fumarate reductase by occupying their quinol binding sites (17, 28). Thus, we presume that quinones directly associated to the terminal oxidases are utilized for electron transfer. In agreement with this assumption HemG was found membrane associated.

Fig. 4.

Model for the E. coli PPO coupled electron transfer reactions and ATP generation. PPO enzymatically converts protógen to proto, probably via hydride transfer to FMN. From this flavin, the electrons are transferred to quinones. Electron uptake from terminal oxidases recycles the quinones. Oxygen is used as electron acceptor via two different cytochrome oxidases. Under anaerobic conditions, electrons are dissipated to the terminal electron acceptors fumarate and nitrate by the respective reductases. Three of the terminal oxidoreductases couple electron transport to protein transfer via the cytoplasmic membrane. The generated proton motive force is employed for ATP generation by ATPase.

A HemG Model.

In absence of structural data for E. coli HemG, computational models can be built by homology modeling using related structures of long chain flavodoxins to which HemG is sequence related (7). The model built in this investigation showed differences compared to the model for E. coli HemG proposed before (8). The main difference was observed for the structure deduced for the long chain insert region which is unique in HemG proteins. This region is supposed to be responsible for specifying both substrate binding (protoporphyrinogen IX and quinones) and membrane anchoring. Our bioinformatic analysis indicated that this region adopts an amphiphilic helical conformation in the predicted structure of E. coli HemG (pdb 2ark) (Fig. 5). The helix is mainly composed of nonpolar residues on one side, while the opposite side is composed of basic charged residues. Such an amphiphilic character may explain the membrane associated feature and the demonstrated ability of HemG to direct electron transfer towards lipophilic quinone molecules via interaction with membrane-bound respiratory complexes.

Fig. 5.

Model structure of E. coli HemG. The structure was generated as outlined in SI Materials and Methods. These analyses predicted the presence of an amphiphilic helix (red) involved in substrate binding, catalysis, and electron transport chain interaction. One side of the helix is mainly composed of nonpolar residues while the opposite side consists of basic charged residues.

Quinone-Dependent Metabolic Reactions.

For a second biosynthetic enzyme, namely class 2 dihydroorotate dehydrogenase, coupling of enzyme catalysis to ubiquinone was described before (29, 30). These FMN containing enzymes of eukaryotes and Gram-negative bacteria are found associated with the cytoplasmatic membrane via an N-terminal extension (31). However, no direct electron transfer to a terminal oxidoreductase was demonstrated for this type of enzyme. A second known example for electron transfer chain coupled process is the formation of disulfide bonds of proteins in the periplasm of Gram-negative bacteria (32). An in vitro system containing DsbA, DsbB, and either cytochrome bd or cytochrome co₃ was employed (33, 34). In contrast to HemG, in this case electrons are transferred from the periplasmic site of the membrane from an enzyme involved in protein modification, not in classical anabolic biosynthesis. Consequently, we showed that an anabolic biosynthetic oxidation is directly coupled to energy conservation via respiration. The biosynthesis of a cofactor is used for ATP generation.

Materials and Methods

Bacterial Strains and Plasmids.

The strains and plasmids used in this work are listed in SI Text Table S1. Growth of the bacterial strains and expression plasmid construction are outlined in SI Materials and Methods.

Recombinant HemG Production in E. Coli and B. Megaterium and Affinity Chromatographic Purification.

E. coli BL21 (λDE3) harboring pETDuet1hemG was used for the recombinant production of His_6X-tagged E. coli HemG in E. coli. Purification of the recombinant protein was achieved by affinity and gel permeation chromatography (SI Materials and Methods).

Production and Purification of Recombinant E. Coli Fumarate, Nitrate, and Cytochrome Oxidoreductases.

The terminal oxidoreductases were recombinantly produced and affinity chromatographically purifed as detailed before (17, 19, 23, 26, 35) with modification described in SI Materials and Methods.

Purification of E. coli PPO from the Membrane Fraction.

The membrane fraction was isolated using sucrose gradient centrifugation and PPO activity enriched by DEAE-Sepharose FF chromatography as outlined in SI Materials and Methods.

Protein Identification Using Proteomics Techniques.

The peptide analysis of the E. coli PPO activity containing fraction after membrane solubilization and chromatographic purification was performed according to standard procedures (36) (SI Materials and Methods). HemG was identified by the presence of at least 17 different HemG specific peptides in a fraction with high PPO activity prepared from E. coli membrane fraction. The identified peptides are listed in Table S2. A HemG containing fraction after affinity chromatographic purification reproducibly of recombinant HemG from cell-free extract yielded the FrdA specific peptides (Table S2). A highly enriched HemG containing fraction prepared from E. coli membranes also contained FrdA, which was identified by various peptides (Table S2).

Protoporphyrinogen IX Oxidase Activity Assays.

The substrate preparation strategy was described by Kushner and coworkers (37). PPO assays were performed and kinetic data obtained as outlined before (38). Additions of quinones and terminal oxidases are detetailed in SI Materials and Methods.

Identification of the HemG Flavin Cofactor.

Spectral analysis and HPLC identification of the HemG FMN cofactor was described before (39) with modifications shown in SI Materials and Methods.