Quantifying structural relationships of metal-binding sites suggests origins of biological electron transfer

Computational exploration of similarities among metal-binding protein structural motifs elucidates the origins of life.

Customized exogenous ferredoxin functions as an efficient electron carrier

Ferredoxin (Fdx) is regarded as the main electron carrier in biological electron transfer and acts as an electron donor in metabolic pathways of many organisms. Here, we screened a self-sufficient P450-derived reductase PRF with promising production yield of 9OHAD (9α-hydroxy4-androstene-3,17-dione) from AD, and further proved the importance of [2Fe–2S] clusters of ferredoxin-oxidoreductase in transferring electrons in steroidal conversion. The results of truncated Fdx domain in all oxidoreductases and mutagenesis data elucidated the indispensable role of [2Fe–2S] clusters in the electron transfer process. By adding the independent plant-type Fdx to the reaction system, the AD (4-androstene-3,17-dione) conversion rate have been significantly improved. A novel efficient electron transfer pathway of PRF + Fdx + KshA (KshA, Rieske-type oxygenase of 3-ketosteroid-9-hydroxylase) in the reaction system rather than KshAB complex system was proposed based on analysis of protein–protein interactions and redox potential measurement. Adding free Fdx created a new conduit for electrons to travel from reductase to oxygenase. This electron transfer pathway provides new insight for the development of efficient exogenous Fdx as an electron carrier. Graphical Abstract

Customized exogenous ferredoxin functions as an efficient electron carrier

Ferredoxin (Fdx) is regarded as the main electron carrier in biological electron transfer and acts as an electron donor in metabolic pathways of many organisms. Here, we screened a self-sufficient P450-derived reductase PRF with promising NADPH reduction activity and 9OHAD production yield and proved the importance of [2Fe-2S] clusters of Fdx-containing oxidoreductase in transferring electrons in steroidal conversion. The truncated Fdx domain in all oxidoreductases, together with mutagenesis data, further elucidated the indispensable role of [2Fe-2S] clusters in the electron transfer process. By adding the independent plant-type Fdx to the reaction system, the AD conversion rate have been significantly improved. A novel efficient electron transfer pathway of PRF+Fdx+KshA in the reaction system rather than KshAB complex system was proposed based on analysis of protein-protein interactions and redox potential measurement. Adding free Fdx created a new conduit for electrons to travel from reductase to oxygenase. This electron transfer pathway provides new insight for the development of efficient exogenous Fdx as an electron carrier.

How to Turn an Electron Transfer Protein into a Redox Enzyme for Biosensing

Cytochrome c is a small globular protein whose main physiological role is to shuttle electrons within the mitochondrial electron transport chain. This protein has been widely investigated, especially as a paradigmatic system for understanding the fundamental aspects of biological electron transfer and protein folding. Nevertheless, cytochrome c can also be endowed with a non-native catalytic activity and be immobilized on an electrode surface for the development of third generation biosensors. Here, an overview is offered of the most significant examples of such a functional transformation, carried out by either point mutation(s) or controlled unfolding. The latter can be induced chemically or upon protein immobilization on hydrophobic self-assembled monolayers. We critically discuss the potential held by these systems as core constituents of amperometric biosensors, along with the issues that need to be addressed to optimize their applicability and response.

Synthesis of redox-active fluorinated 5-hydroxytryptophans as molecular reporters for biological electron transfer

Chemoenzymatic synthesis of fluorinated 5-hydroxytryptophans provides a novel set of non-canonical tryptophan amino acids useful for probes of biological electron transfer reactions.

Shallow Distance Dependence for Proton-Coupled Tyrosine Oxidation in Oligoproline Peptides

We have explored the kinetic effect of increasing electron transfer distance in a biomimetic, proton coupled electron transfer system (PCET). Biological electron transfer is often simultaneous with proton transfer in order to avoid the high-energy, charged intermediates resulting from the stepwise transfer of protons and electrons. These concerted proton electron transfer (CPET) reactions are implicated in numerous biological electron transfer pathways. In many cases, proton transfer is coupled to long-range electron transfer. While many studies have shown that the rate of electron transfer is sensitive to the distance between the electron donor and acceptor, extensions to biological CPET reactions are sparse. The possibility of a unique electron transfer distance dependence for CPET reactions deserves further exploration, as this could have implications for how we understand biological electron transfer. We therefore explored the electron transfer distance dependence for the CPET oxidation of tyrosine in a model system. We prepared a series of metallopeptides with a tyrosine separated from a Ru(bpy)₃²⁺ complex by an oligoproline bridge of increasing length. Rate constants for intramolecular tyrosine oxidation were measured using the flash-quench transient absorption technique in aqueous solutions. The rate constants for tyrosine oxidation decreased by 125-fold with three added prolines residues between tyrosine and the oxidant. By comparison, related intramolecular ET rate constants in very similar constructs were reported to decrease by 4-5 orders of magnitude over the same number of prolines. The observed shallow distance dependence for tyrosine oxidation is proposed to originate, at least in part, from the requirement for stronger oxidants, leading to a smaller hole transfer tunneling barrier height. The shallow distance dependence observed here and extensions to distance dependent CPET reactions have far-reaching implications for long-range charge transfers

Shallow Distance Dependence for Proton-Coupled Tyrosine Oxidation in Oligoproline Peptides

We have explored the kinetic effect of increasing electron transfer distance in a biomimetic, proton coupled electron transfer system (PCET). Biological electron transfer is often simultaneous with proton transfer in order to avoid the high-energy, charged intermediates resulting from the stepwise transfer of protons and electrons. These concerted proton electron transfer (CPET) reactions are implicated in numerous biological electron transfer pathways. In many cases, proton transfer is coupled to long-range electron transfer. While many studies have shown that the rate of electron transfer is sensitive to the distance between the electron donor and acceptor, extensions to biological CPET reactions are sparse. The possibility of a unique electron transfer distance dependence for CPET reactions deserves further exploration, as this could have implications for how we understand biological electron transfer. We therefore explored the electron transfer distance dependence for the CPET oxidation of tyrosine in a model system. We prepared a series of metallopeptides with a tyrosine separated from a Ru(bpy)₃²⁺ complex by an oligoproline bridge of increasing length. Rate constants for intramolecular tyrosine oxidation were measured using the flash-quench transient absorption technique in aqueous solutions. The rate constants for tyrosine oxidation decreased by 125-fold with three added prolines residues between tyrosine and the oxidant. By comparison, related intramolecular ET rate constants in very similar constructs were reported to decrease by 4-5 orders of magnitude over the same number of prolines. The observed shallow distance dependence for tyrosine oxidation is proposed to originate, at least in part, from the requirement for stronger oxidants, leading to a smaller hole transfer tunneling barrier height. The shallow distance dependence observed here and extensions to distance dependent CPET reactions have far-reaching implications for long-range charge transfers