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Heme Biosynthesis Is Coupled to Electron Transport Chains for Energy Generation

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Heme Biosynthesis Is Coupled to Electron Transport Chains for Energy Generation Heme biosynthesis is coupled to electron transport chains for energy generation Kalle Möbiusa, Rodrigo Arias-Cartinb, Daniela Breckaua, Anna-Lena Hänniga, Katrin Riedmannc, Rebekka Biedendiecka, Susanne Schrödera, Dörte Becherd, Axel Magalonb, Jürgen Mosera, Martina Jahna, and Dieter Jahna,1 aInstitute of Microbiology, University Braunschweig, Brunswick, Germany; bLaboratoire de Chimie Bactérienne, Institute de Microbiologie de la Méditerraneé, Centre National de la Recherche Scientifique, Marseille, France; cDepartment of Agrarcultural Technology, Johann Heinrich von Thünen-Institut, Brunswick, Germany; and dInstitute of Microbiology, University of Greifswald, Germany Edited* by Dieter Söll, Yale University, New Haven, CT, and approved April 23, 2010 (received for review January 30, 2010) Cellular energy generation uses membrane-localized electron O2 tensions in the growth medium, cytochrome bo3 (EC 1.10.3; transfer chains for ATP synthesis. Formed ATP in turn is consumed Cyo) dominates over all other terminal oxidases. At low O2 ten- for the biosynthesis of cellular building blocks. In contrast, heme sions the second cytochrome oxidase, termed bd (EC 1.10.3; cofactor biosynthesis was found driving ATP generation via Cyd), is present in the cytoplasmic membrane (10–15). Both electron transport after initial ATP consumption. The FMN enzyme enzyme systems are known to couple the four-electron reduction protoporphyrinogen IX oxidase (HemG) of Escherichia coli of O2 to two H2O molecules with the formation of a proton po- abstracts six electrons from its substrate and transfers them via tential via the membrane (13, 14). When O2 is absent, E. coli is bo bd ubiquinone, cytochrome 3 (Cyo) and cytochrome (Cyd) capable of utilizing nitrate, nitrite, TMAO (trimethylamine N- oxidase to oxygen. Under anaerobic conditions electrons are trans- oxide), DMSO (dimethylsulfoxide), and fumarate as alternative ferred via menaquinone, fumarate (Frd) and nitrate reductase terminal electron acceptors (9). The dissimilatory nitrate reduc- (Nar). Cyo, Cyd and Nar contribute to the proton motive force that tase (EC 1.6.6.1) NarGHI is a membrane-bound enzyme complex drives ATP formation. Four electron transport chains from HemG consisting of the nitrate-reducing subunit NarG, the electron- via diverse quinones to Cyo, Cyd, Nar, and Frd were reconstituted transfer subunit NarH, and the quinol-oxidizing subunit NarI in vitro from purified components. Characterization of E. coli mu- (15–18). Fumarate reductase (EC 1.3.1.6; Frd) of E. coli is a tants deficient in nar, frd, cyo, cyd provided in vivo evidence for a membrane-bound flavoprotein catalyzing the cytoplasmic reduc- BIOCHEMISTRY detailed model of heme biosynthesis coupled energy generation. tion of fumarate to succinate, as well as the oxidation of quinols in the membrane (9, 19). The biochemical principles of coupling anabolism coupled catabolism ∣ protoporphyrinogen IX oxidase ∣ HemG ∣ protoporphyrinogen IX oxidation in E. coli to the outlined tetrapyrrole ∣ respiration electron transport chains were completely unknown. Results and Discussion eme is an essential cofactor of enzymes in electron transport Hchain mediated energy generation. It is synthesized using a E. coli HemG Is a Membrane Associated Protoporphyrinogen IX Oxi- highly conserved pathway (1). The penultimate step of heme bio- dase. In order to purify E. coli PPO we first established an enzyme synthesis—the conversion of protoporphyrinogen IX (proto’gen) assay using PPO active cell-free extract. However, this assay via the abstraction of six electrons into protoporphyrin IX (proto) always required the presence of fumarate reductase and mena- μ —is catalyzed by protoporphyrinogen IX oxidases (PPO; EC quinone for PPO activity. Hence, we replaced those with 2.5 M 1.1.3.4). An O2-dependent PPO, usually encoded by the hemY of the artificial electron acceptor triphenyltetrazolium chloride gene, is missing in Escherichia coli (2). Over 30 yr ago an oxy- (TTC, Fig. 1). In this context several of the tested potential elec- gen-independent PPO activity was detected in cell-free extracts tron acceptors [i.e. 2, 6-dichloroindophenol (DCIP), phenazin- of E. coli which was dependent on electron transfer to nitrate methosulfate (PMS), menadione, and vitamin K1, respectively] or fumarate (3–5). The observation indicated the coupling of were found to directly oxidize the substrate in the absence of anabolic heme biosynthesis to catabolic electron chain-driven HemG and were therefore not used in the activity assay system. ATP synthesis. Complementation of the proto accumulating TTC was the only stable electron acceptor utilized by HemG and E. coli strain SASX38 yielded the hemG gene (6). Because of consequently employed in this study. Corresponding chemical M ¼ 21;200 structures are given in Fig. 1. Using the TTC-based enzyme test the small size of the deduced HemG protein ( r ) 247 ∕ ∕ and the fact that no PPO activity has been shown for the isolated system, PPO activity of pmol proto mg protein h was found peptide, it was assumed that HemG may be a subunit of a larger associated with an isolated membrane fraction of E. coli (Table S1). Crude cell-free extract (53 pmol proto∕mg protein∕ PPO complex (2). Additionally, HemG does not share any amino 11 ∕ ∕ acid sequence homology to oxygen-dependent HemY. Very re- h) and the cytosolic fraction ( pmol proto mg protein h) were cently it was reported that purified recombinant E. coli HemG significantly less active. Membrane fraction proteins were solu- carries menadione-dependent protoporphyrinogen IX oxidase bilized using the detergent Thesit® (2-dodecoxyethanol) and activity (7). The recombinant protein was described as completely further purified via anion exchange chromatography. Obtained water soluble. A structural model based on the homology to long chain flavodoxins was proposed (7). Respiratory reactions possi- Author contributions: K.M., R.A.-C., R.B., S.S., A.M., J.M., and M.J. designed research; bly coupled to the HemG reaction are usually used to drive mem- K.M., R.A.-C., D. Breckau, A.-L.H., K.R., R.B., S.S., D. Becher, A.M., J.M., M.J., and D.J. brane-bound electron transport chain for the formation of an performed research; K.M., R.A.-C., R.B., S.S., D. Becher, and M.J. contributed new reagents/analytic tools; K.M., D. Breckau, R.B., S.S., D. Becher, A.M., M.J., and D.J. analyzed electrochemical ion gradient. This membrane-localized gradient, data; and K.M., A.M., M.J., and D.J. wrote the paper. called proton motive force, provides the energy for ATPase to The authors declare no conflict of interest. generate ATP from ADP and Pi (8, 9). Electron transport chains *This Direct Submission article had a prearranged editor. consist of numerous primary dehydrogenases and quinone-linked 1To whom correspondence should be addressed at: Institute of Microbiology, University terminal oxidoreductases. Primary dehydrogenases in E. coli in- Braunschweig, Spielmannstrasse 7, D-38106 Braunschweig, Germany. E-mail: d.jahn@ clude those for NADH, lactate, glucose, formate, and hydrogen tu-bs.de. (9). The three quinones of E. coli are ubiquinone (UQ), mena- This article contains supporting information online at www.pnas.org/lookup/suppl/ quinone (MQ), and demethylmenaquinone (DMQ) (9). At high doi:10.1073/pnas.1000956107/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1000956107 PNAS Early Edition ∣ 1of6 Downloaded by guest on September 25, 2021 O Cl N N + SO 2- Na+ - 4 O O N Cl O Menadione (Provitamin K3) 2, 6- dichloroindophenol (DCIP) Phenazinmethasulfate (PMS) o O CH3 o CH CH CH 3 3 3 O Fig. 1. Electron acceptors tested for E. coli HemG. The structures of the following electron acceptors used in this Phylloquinone (Vitamin K1) Menaquinone (Vitamin K1) work are depicted: 2, 6-dichloroindolphenol (DCIP) with 00 00 an E of þ237 mV, menadione (provitamin K3, E ¼ 0 −205 mV), phenazinmethosulfate (PMS, E0 ¼þ80 mV), 0 O 2, 6-triphenoltetrazoliumchloride (TTC, E0 ¼ −80 mV), CH 00 N CH O 3 phylloquinone (vitamin K1, E ¼ −170 mV), menaqui- N 3 00 none (vitamin K2, E ¼ −74 mV), and ubiquinone (coen- N N CH 00 00 + 3 O H zyme A, E ¼þ110 mV). HemG cofactor FMN has E ¼ Cl- O CH −190 3 6 - 10 mV. Redox potentials are given for the acceptor- donor couples of the free compounds (40, 41). Protein environments might individually change the correspond- Ubiquinone (Coenzyme Q) 2, 6 -triphenoltetrazoliumchloride (TTC) ing values. fractions harboring the highest specific PPO activity were ana- lyzed for the identity of contained proteins by mass spectrometry. The most frequently detected peptides in the analyzed samples A rel M W1 W2 E1 E2 belonged to the HemG protein (Table S2). Nevertheless, the MW W3 question still remained whether the strong PPO activity of the purified membrane fraction is caused by HemG alone or whether 70 the membrane fraction contained other components contributing 55 to the 6 e− oxidation of proto’gen. The B. megaterium genome 45 does neither possess a hemG gene nor the enzymes of anaerobic 35 respiration including fumarate and nitrate reductase. Bacilli are synthesizing proto using the oxygen-dependent flavin-enzyme 25 HemY (20). In order to exclude association of E. coli HemG with HemG additional potential PPO subunits from E. coli the HemG protein 15 was produced fused to a His-tag in B. megaterium (Fig. 2). With TTC as electron acceptor B. megaterium cell-free extract contain- ing recombinant E. coli, HemG revealed the formation of 10 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 300 enzyme activity in the presence of TTC as electron acceptor B (23 nmol proto∕mg protein∕h). Removal of the fused His-tag 250 via protease digestion did not change the catalytic properties, neither with TTC (21 nmol proto∕mg protein∕h) nor with puri- 200 fied fumarate reductase as electron acceptors (55 nmol proto∕ mg protein∕h).
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