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Abstract The core metabolic reactions of life drive electrons through a class of redox protein enzymes, the oxidoreductases. The energetics of electron flow is determined by the redox potentials of organic and inorganic cofactors as tuned by the protein environment. Understanding how protein structure affects oxidation–reduction energetics is crucial for studying metabolism, creating bioelectronic systems, and tracing the history of biological energy utilization on Earth. We constructed ProtReDox (https://protein-redox-potential.web.app), a manually curated database of experimentally determined redox potentials. With over 500 measurements, we can begin to identify how proteins modulate oxidation–reduction energetics across the tree of life. By mapping redox potentials onto networks of oxidoreductase fold evolution, we can infer the evolution of electron transfer energetics over deep time. ProtReDox is designed to include user‐contributed submissions with the intention of making it a valuable resource for researchers in this field. 

It has long been known that the alteration of protein side chains that occlude or expose the heme cofactor to water can greatly affect the stability of the oxyferrous heme state. Here, we demonstrate that the rate of dynamically driven water penetration into the core of an artificial oxygen transport protein also correlates with oxyferrous state lifetime by reducing global dynamics, without altering the structure of the active site, via the simple linking of the two monomers in a homodimeric artificial oxygen transport protein using a glycine-rich loop. The tethering of these two helices does not significantly affect the active site structure, pentacoordinate heme-binding affinity, reduction potential, or gaseous ligand affinity. It does, however, significantly reduce the hydration of the protein core, as demonstrated by resonance Raman spectroscopy, backbone amide hydrogen exchange, and pKa shifts in buried histidine side chains. This further destabilizes the charge-buried entatic state and nearly triples the oxyferrous state lifetime. These data are the first direct evidence that dynamically driven water penetration is a rate-limiting step in the oxidation of these complexes. It furthermore demonstrates that structural rigidity that limits water penetration is a critical design feature in metalloenzyme construction and provides an explanation for both the failures and successes of earlier attempts to create oxygen-binding proteins. 

Ancestral metabolic processes involve the reversible oxidation of molecular hydrogen by hydrogenase. Extant hydrogenase enzymes are complex, comprising hundreds of amino acids and multiple cofactors. We designed a 13–amino acid nickel-binding peptide capable of robustly producing molecular hydrogen from protons under a wide variety of conditions. The peptide forms a di-nickel cluster structurally analogous to a Ni-Fe cluster in [NiFe] hydrogenase and the Ni-Ni cluster in acetyl-CoA synthase, two ancient, extant proteins central to metabolism. These experimental results demonstrate that modern enzymes, despite their enormous complexity, likely evolved from simple peptide precursors on early Earth.