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Despite the current availability of several crystal structures of purple acid phosphatases, to date there is no direct evidence for solvent-derived ligands occupying terminal positions in the active enzyme. This is of central importance, because catalysis has been shown to proceed through the direct attack on a metal-bound phosphate ester by a metal-activated solvent-derived moiety, which has been proposed to be either (i) a hydroxide ligand terminally bound to the ferric center or (ii) a bridging hydroxide. In this work we use (2)H Q-band (35 GHz) pulsed electron-nuclear double resonance (ENDOR) spectroscopy to identify solvent molecules coordinated to the active mixed-valence (Fe(3+)Fe(2+)) form of the dimetal center of uteroferrin (Uf), as well as to its complexes with the anions MoO(4), AsO(4), and PO(4). The solvent-derived coordination of the dinuclear center of Uf as deduced from ENDOR data includes a bridging hydroxide and a terminal water/hydroxide bound to Fe(2+) but no terminal water/hydroxide bound to Fe(3+). The terminal water is lost upon anion binding while the hydroxyl bridge remains. These results are not compatible with a hydrolysis mechanism involving a terminal Fe(3+)-bound nucleophile, but they are consistent with a mechanism that relies on the bridging hydroxide as the nucleophile.

Twisted stalks are organo-mineral structures produced by some microaerophilic Fe(II)-oxidizing bacteria at O 2 concentrations as low as 3 mM. The presence of these structures in rocks having experienced a diagenetic history could indicate microbial Fe(II)-oxidizing activity as well as localized abundance of oxygen at the time of sediment deposition. Here we use spectroscopy and analytical microscopy to evaluate if-and what kind of-transformations occur in twisted stalks through experimental diagenesis. Unique mineral textures appear on stalks as temperature and pressure conditions increase. Haematite and magnetite form from ferrihydrite at 170°C-120 MPa. Yet the twisted morphology of the stalks, and the organic matrix, mainly composed of long-chain saturated aliphatic compounds, are preserved at 250°C-140 MPa. Our results suggest that iron minerals might play a role in maintaining the structural and chemical integrity of stalks under diagenetic conditions and provide spectroscopic signatures for the search of ancient life in the rock record.

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