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 bo(3) (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.

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).

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.

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.