Ferredoxin reduction by hydrogen with iron functions as an evolutionary precursor of flavin-based electron bifurcation

Abstract

Autotrophic theories for the origin of metabolism posit that the first cells satisfied their carbon needs from CO₂ and were chemolithoautotrophs that obtained their energy and electrons from H₂. The acetyl-CoA pathway of CO₂ fixation is central to that view because of its antiquity: Among known CO₂ fixing pathways it is the only one that is i) exergonic, ii) occurs in both bacteria and archaea, and iii) can be functionally replaced in full by single transition metal catalysts in vitro. In order to operate in cells at a pH close to 7, however, the acetyl-CoA pathway requires complex multi-enzyme systems capable of flavin-based electron bifurcation that reduce low potential ferredoxin-the physiological donor of electrons in the acetyl-CoA pathway-with electrons from H₂. How can the acetyl-CoA pathway be primordial if it requires flavin-based electron bifurcation? Here, we show that native iron (Fe⁰), but not Ni⁰, Co⁰, Mo⁰, NiFe, Ni₂Fe, Ni₃Fe, or Fe₃O₄, promotes the H₂-dependent reduction of aqueous Clostridium pasteurianum ferredoxin at pH 8.5 or higher within a few hours at 40 °C, providing the physiological function of flavin-based electron bifurcation, but without the help of enzymes or organic redox cofactors. H₂-dependent ferredoxin reduction by iron ties primordial ferredoxin reduction and early metabolic evolution to a chemical process in the Earth's crust promoted by solid-state iron, a metal that is still deposited in serpentinizing hydrothermal vents today.

Keywords: acetyl CoA pathway; origin of life; origin of metabolism; serpentinization; transition metals.

Fig. 1.

Ferredoxin is the primordial one-electron carrier in metabolism. (A) Flavin-based electron bifurcation is required to reduce low potential ferredoxin for CO₂ fixation using electrons from H₂ (schematic). The flavoprotein and its high potential acceptor varies across organisms and pathways (–42). (B) The structure of C. pasteurianum ferredoxin (PDB ID: 1CLF). Other renderings of ferredoxin are given in SI Appendix, Fig. S1. (C) The structure of the two forms of hydrogenase used to oxidize H₂ for Fd reduction and their catalytic metal clusters compared to Fd from C. pasteurianum. The structures are for MvhAGD, the [NiFe] hydrogenase from Methanothermococcus thermolithotrophicus (PDB ID: 5ODC) (36) and HydABC, the [FeFe] hydrogenase from Acetobacterium woodii (PDB ID: 8A5E) (37). The CN^– and aminodithiolate ligands are missing in the structure of the H-cluster of HydABC. One protomer of HydABC and one protomer of MvhAGD are shown, the biologically active complexes being a homodimer of heterotrimers and a homodimer of heterohexamers (including the heterodisulfide reductase), respectively. Under nickel limitation, some methanogens express a third form of hydrogenase with a unique iron-guanylylpyrinidol cofactor that transfers electrons from H₂ directly to methenyl-tetrahydromethanopterin without FeS cluster intermediates (8).

Fig. 2.

Photometric assay of Fd reduction. (A) UV-Vis spectra following the oxidation of 30 µM of reduced Fd from C. pasteurianum in 0.133 M phosphate buffer at pH 8.5 through air exposure for 300 s. Fd was previously reduced through addition of two equivalents of sodium dithionite (Na₂S₂O₄). (B) Magnified boxed region in A showing the difference in absorption of the [4Fe4S] clusters at 420 nm and the isosbestic point at 354 nm (56), used for normalization. The time course of Fd reduction is shown in SI Appendix, Fig. S2.

Fig. 3.

Metals tested for H₂-dependent Fd reduction. Reaction of 30 µM Fd from C. pasteurianum with 0.1 mmol (A) native metal and (B) metal alloys and magnetite under a hydrogen atmosphere [5 bar] in 0.133 M phosphate buffer at pH 8.5. Normalized absorption of [4Fe4S] clusters in Fd is shown (Methods). Raw data for iron shown in SI Appendix, Fig. S3.

Fig. 4.

Time course of Fe⁰-promoted H₂-dependent Fd reduction. The lag time of about 1 h might reflect delayed formation of chemisorbed hydrogen on the iron surface (61, 62) or H₂-dependent formation of active sites on the metal surface. A similar lag time was observed for H₂-dependent NAD⁺ reduction on Fe⁰ (57).

Fig. 5.

H₂-dependent Fd reduction at pH 10. Reaction of 30 µM Fd from C. pasteurianum with 0.1 mmol metal. (A) Native iron and (B) native cobalt and native nickel under a hydrogen atmosphere [5 bar] in 0.133 M phosphate buffer at pH 8.5 and pH 10. Normalized absorption of [4Fe4S] clusters in Fd is shown (Methods).

Fig. 6.

pH dependence of Fd reduction with H₂. Reaction of 30 µM Fd from C. pasteurianum with 0.1 mmol native iron under a hydrogen atmosphere [5 bar] in 0.133 M and 1.33 M phosphate buffer at pH 8.5. Normalized absorption of [4Fe4S] clusters in Fd is shown (Methods). The midpoint potential of H₂ was calculated using the Nernst equation.

Fig. 7.

Three phases in the evolution of autotrophy via the acetyl-CoA pathway. (A) Metal-catalyzed pyruvate synthesis from H₂ and CO₂ (–20, 80) as the ancestral state of the acetyl-CoA pathway. (B) Iron-dependent ferredoxin reduction with H₂ as a source of the physiological reductant (reduced ferredoxin) for CODH/ACS (5, 7, 60) and PFOR (6, 34, 60) in an intermediate stage of physiological evolution before the origin of flavin-based electron bifurcation. (C) H₂ oxidation via Fe–Fe and Ni–Fe hydrogenases and flavin-based electron bifurcation for the synthesis of reduced ferredoxin in a fully soluble enzymatic system encoded by genes. The source of methyl groups for the ACS reaction in (B) can either be geochemical (72, 83) as in (A) or biochemical from CO₂ as in the extant acetyl-CoA pathway (2).