Journal
BIOCHIMICA ET BIOPHYSICA ACTA-BIOENERGETICS
Volume 1857, Issue 9, Pages 1353-1362Publisher
ELSEVIER
DOI: 10.1016/j.bbabio.2016.05.003
Keywords
Arsenite oxidase; Molybdenum enzyme; Optical spectroscopy; EPR spectroscopy; Redox titrations
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Funding
- CNRS
- CEA
- Aix-Marseille University
- ANR [11-BSV5-005-01]
- NASA Astrobiology Institute (Icy Worlds)
- French EPR network (RENARD) [IR3443]
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While the molybdenum cofactor in the majority of bisPGD enzymes goes through two consecutive 1-electron redox transitions, previous protein-film voltammetric results indicated the possibility of cooperative (n = 2) redox behavior in the bioenergetic enzyme arsenite oxidase (Aio). Combining equilibrium redox titrations, optical and EPR spectroscopies on concentrated samples obtained via heterologous expression, we unambiguously confirm this claim and quantify Aio's redox cooperativity. The stability constant, K-s of the Mo-v semi-reduced intermediate is found to be lower than 10(-3). Site-directed mutagenesis of residues in the vicinity of the Mo-cofactor demonstrates that the degree of redox cooperativity is sensitive to H-bonding interactions between the pyranopterin moieties and amino acid residues. Remarkably, in particular replacing the Gln-726 residue by Gly results in stabilization of (low-temperature) EPR-observable Mo-v with K-s = 4. As evidenced by comparison of room temperature optical and low temperature EPR titrations, the degree of stabilization is temperature dependent. This highlights the importance of room-temperature redox characterizations for correctly interpreting catalytic properties in this group of enzymes. Geochemical and phylogenetic data strongly indicate that molybdenum played an essential biocatalytic roles in early life. Molybdenum's redox versatility and in particular the ability to show cooperative (n = 2) redox behavior provide a rationale for its paramount catalytic importance throughout the evolutionary history of life. Implications of the H-bonding network modulating Molybdenum's redox properties on details of a putative inorganic metabolism at life's origin are discussed. (C) 2016 Published by Elsevier B.V.
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