SignificanceRibosomes are found in every living organism, where they are responsible for the translation of messenger RNA into protein. The ribosome’s centrality to cell function is underscored by its evolutionary conservation; the core structure has changed little since its inception ∼4 billion years ago when ecosystems were anoxic and metal-rich. The ribosome is a model system for the study of bioinorganic chemistry, owing to the many highly coordinated divalent metal cations that are essential to its function. We studied the structure, function, and cation content of the ribosome under early Earth conditions (low O2, high Fe2+, and high Mn2+). Our results expand the roles of Fe2+ and Mn2+ in ancient and extant biochemistry as cofactors for ribosomal structure and function.

Reactive Fe(III) minerals can influence methane (CH ) emissions by inhibiting microbial methanogenesis or by stimulating anaerobic CH oxidation. The balance between Fe(III) reduction, methanogenesis, and CH oxidation in ferruginous Archean and Paleoproterozoic oceans would have controlled CH fluxes to the atmosphere, thereby regulating the capacity for CH to warm the early Earth under the Faint Young Sun. We studied CH and Fe cycling in anoxic incubations of ferruginous sediment from the ancient ocean analogue Lake Matano, Indonesia, over three successive transfers (500 days in total). Iron reduction, methanogenesis, CH oxidation, and microbial taxonomy were monitored in treatments amended with ferrihydrite or goethite. After three dilutions, Fe(III) reduction persisted only in bottles with ferrihydrite. Enhanced CH production was observed in the presence of goethite, highlighting the potential for reactive Fe(III) oxides to inhibit methanogenesis. Supplementing the media with hydrogen, nickel and selenium did not stimulate methanogenesis. There was limited evidence for Fe(III)-dependent CH oxidation, although some incubations displayed CH -stimulated Fe(III) reduction. 16S rRNA profiles continuously changed over the course of enrichment, with ultimate dominance of unclassified members of the order Desulfuromonadales in all treatments. Microbial diversity decreased markedly over the course of incubation, with subtle differences between ferrihydrite and goethite amendments. These results suggest that Fe(III) oxide mineralogy and availability of electron donors could have led to spatial separation of Fe(III)-reducing and methanogenic microbial communities in ferruginous marine sediments, potentially explaining the persistence of CH as a greenhouse gas throughout the first half of Earth history.

Electroactive type IV pili, or e-pili, are used by some microbial species for extracellular electron transfer. Recent studies suggest that e-pili may be more phylogenetically and structurally diverse than previously assumed. Here, we used updated aromatic density thresholds (≥9.8% aromatic amino acids, ≤22-aa aromatic gaps and aromatic amino acids at residues 1, 24, 27, 50 and/or 51, and 32 and/or 57) to search for putative e-pilin genes in metagenomes from diverse ecosystems with active microbial metal cycling. Environmental putative e-pilins were diverse in length and phylogeny, and included truncated e-pilins in Geobacter spp., as well as longer putative e-pilins in Fe(II)oxidizing Betaproteobacteria and Zetaproteobacteria.

Divalent metal cations are essential to the structure and function of the ribosome. Previous characterizations of the ribosome performed under standard laboratory conditions have implicated Mg2+ as a primary mediator of ribosomal structure and function. Possible contributions of Fe2+ as a ribosomal cofactor have been largely overlooked, despite the ribosome's early evolution in a high Fe2+ environment, and the continued use of Fe2+ by obligate anaerobes inhabiting high Fe2+ niches. Here, we show that (i) Fe2+ cleaves RNA by in-line cleavage, a non-oxidative mechanism that has not previously been shown experimentally for this metal, (ii) the first-order in-line rate constant with respect to divalent cations is >200 times greater with Fe2+ than with Mg2+, (iii) functional ribosomes are associated with Fe2+ after purification from cells grown under low O2 and high Fe2+ and (iv) a small fraction of Fe2+ that is associated with the ribosome is not exchangeable with surrounding divalent cations, presumably because those ions are tightly coordinated by rRNA and deeply buried in the ribosome. In total, these results expand the ancient role of iron in biochemistry and highlight a possible new mechanism of iron toxicity.

15In extant biochemistry, Mg 2+ ions are essential for both structure and function of the ribosome (4,5) and for 16functions of many enzymes involved in translation (6). The translation system, which synthesizes all coded 17 protein (7), originated and matured during the Archean Eon (4-2.5 Ga; (8)). The common core of the 18 ribosome and many other aspects of the translation system have remained essentially frozen since the last 19 universal common ancestor (9,10). 20In ribosomes, structural Mg 2+ ions occur in rRNA-Mg 2+ clamps (11) (Fig. 1a), in dinuclear Mg 2+ -21 microclusters that frame the peptidyl transferase center (12) (Fig. 1b), and at the SSU-LSU interface (13) 22( Fig. 1c). Functional Mg 2+ ions stabilize a critical bend in mRNA between the P-site and A-site codons (14) 23 (Fig. 1d), and mediate rRNA-tRNA/mRNA interactions (15) (Fig. 1e, f 26All rights reserved. No reuse allowed without permission.(which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. (which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. 97RO). Thus, a total of 2.5 mM "background" Mg 2+ was present in each reaction (Fig. S1) MgCl2 and FeCl2 which were tested over a range of final concentrations (1, 3, 4, 5, 6, 7, 8, 9, and 11 mM; 122 All rights reserved. No reuse allowed without permission.(which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. the amounts of metals in our assay reaction, which had no effect on DHFR activity (data not shown). 136Ribosome iron content. In order to measure the iron content of ribosomes, a 300 µL reaction was prepared 137 containing 45 µL of a 13.3 µM stock of E. coli ribosomes (New England Biolabs catalog # P0763S), 7 mM