Characterization of ferredoxins involved in electron transfer pathways for nitrogen fixation implicates differences in electronic structure in tuning 2[4Fe4S] Fd activity.

Ferredoxins (Fds) are small proteins which shuttle electrons to pathways like biological nitrogen fixation. Physical properties tune the reactivity of Fds with different pathways, but knowledge on how these properties can be manipulated to engineer new electron transfer pathways is lacking. Recently, we showed that an evolved strain of Rhodopseudomonas palustris uses a new electron transfer pathway for nitrogen fixation.

Rapid-reaction kinetics of the bifurcating NAD-dependent NADPH:ferredoxin oxidoreductase NfnI from Pyrococcus furiosus.

We have investigated the kinetics of NAD-dependent NADPH:ferredoxin oxidoreductase (NfnI), a bifurcating transhydrogenase that takes two electron pairs from NADPH to reduce two ferredoxins and one NAD through successive bifurcation events. NADPH reduction takes place at the bifurcating FAD of NfnI's large subunit, with high-potential electrons transferred to the [2Fe-2S] cluster and S-FADH of the small subunit, ultimately on to NAD; low-potential electrons are transferred to two [4Fe-4S] clusters of the large subunit and on to ferredoxin. Reduction of NfnI by NADPH goes to completion only at higher pH, with a limiting k of 36 ± 1.

Structural and biophysical properties of a [4Fe4S] ferredoxin-like protein from Synechocystis sp. PCC 6803 with a unique two domain structure.

Electron carrier proteins (ECPs), binding iron-sulfur clusters, are vital components within the intricate network of metabolic and photosynthetic reactions. They play a crucial role in the distribution of reducing equivalents. In Synechocystis sp.

Spectral deconvolution of electron-bifurcating flavoproteins.

Electron-bifurcating flavoproteins catalyze the tightly coupled reduction of high- and low-potential acceptors using a median-potential electron donor, and are invariably complex systems with multiple redox-active centers in two or more subunits. Methods are described that permit, in favorable cases, the deconvolution of spectral changes associated with reduction of specific centers, making it possible to dissect the overall process of electron bifurcation into individual, discrete steps.

Correction: The influence of electron utilization pathways on photosystem I photochemistry in sp. PCC 6803.

[This corrects the article DOI: 10.1039/D2RA01295B.].

Site-Differentiated Iron-Sulfur Cluster Ligation Affects Flavin-Based Electron Bifurcation Activity.

Electron bifurcation is an elegant mechanism of biological energy conversion that effectively couples three different physiologically relevant substrates. As such, enzymes that perform this function often play critical roles in modulating cellular redox metabolism. One such enzyme is NADH-dependent reduced-ferredoxin: NADP oxidoreductase (NfnSL), which couples the thermodynamically favorable reduction of NAD to drive the unfavorable reduction of ferredoxin from NADPH.

The pathway for coenzyme M biosynthesis in bacteria.

Mercaptoethane sulfonate or coenzyme M (CoM) is the smallest known organic cofactor and is most commonly associated with the methane-forming step in all methanogenic archaea but is also associated with the anaerobic oxidation of methane to CO in anaerobic methanotrophic archaea and the oxidation of short-chain alkanes in species. It has also been found in a small number of bacteria capable of the metabolism of small organics. Although many of the steps for CoM biosynthesis in methanogenic archaea have been elucidated, a complete pathway for the biosynthesis of CoM in archaea or bacteria has not been reported.

The influence of electron utilization pathways on photosystem I photochemistry in sp. PCC 6803.

The capacity of cyanobacteria to adapt to highly dynamic photon flux and nutrient availability conditions results from controlled management and use of reducing power, and is a major contributing factor to the efficiency of photosynthesis in aquatic environments. The response to changing conditions includes modulating gene expression and protein-protein interactions that serve to adjust the use of electron flux and mechanisms that control photosynthetic electron transport (PET). In this regard, the photochemical activity of photosystem I (PSI) reaction centers can support balancing of cyclic (CEF) and linear electron flow (LEF), and the coupling of redox carriers for use by electron utilization pathways.

A site-differentiated [4Fe-4S] cluster controls electron transfer reactivity of [FeFe]-hydrogenase I.

One of the many functions of reduction-oxidation (redox) cofactors is to mediate electron transfer in biological enzymes catalyzing redox-based chemical transformation reactions. There are numerous examples of enzymes that utilize redox cofactors to form electron transfer relays to connect catalytic sites to external electron donors and acceptors. The compositions of relays are diverse and tune transfer thermodynamics and kinetics towards the chemical reactivity of the enzyme.

An uncharacteristically low-potential flavin governs the energy landscape of electron bifurcation.

Electron bifurcation, an energy-conserving process utilized extensively throughout all domains of life, represents an elegant means of generating high-energy products from substrates with less reducing potential. The coordinated coupling of exergonic and endergonic reactions has been shown to operate over an electrochemical potential of ∼1.3 V through the activity of a unique flavin cofactor in the enzyme NADH-dependent ferredoxin-NADP+ oxidoreductase I.

The role of thermodynamic features on the functional activity of electron bifurcating enzymes.

Electron bifurcation is a biological mechanism to drive a thermodynamically unfavorable redox reaction through direct coupling with an exergonic reaction. This process allows microorganisms to generate high energy reducing equivalents in order to sustain life and is often found in anaerobic metabolism, where the energy economy of the cell is poor. Recent work has revealed details of the redox energy landscapes for a variety of electron bifurcating enzymes, greatly expanding the understanding of how energy is transformed by this unique mechanism.

The structure and reactivity of the HoxEFU complex from the cyanobacterium sp. PCC 6803.

Cyanobacterial Hox is a [NiFe] hydrogenase that consists of the hydrogen (H)-activating subunits HoxYH, which form a complex with the HoxEFU assembly to mediate reactions with soluble electron carriers like NAD(P)H and ferredoxin (Fdx), thereby coupling photosynthetic electron transfer to energy-transforming catalytic reactions. Researchers studying the HoxEFUYH complex have observed that HoxEFU can be isolated independently of HoxYH, leading to the hypothesis that HoxEFU is a distinct functional subcomplex rather than an artifact of Hox complex isolation. Moreover, outstanding questions about the reactivity of Hox with natural substrates and the site(s) of substrate interactions and coupling of H, NAD(P)H, and Fdx remain to be resolved.

Electron bifurcation: progress and grand challenges.

Electron bifurcation moves electrons from a two-electron donor to reduce two spatially separated one-electron acceptors. If one of the electrons reduces a high-potential (lower energy) acceptor, then the other electron may proceed "uphill" to reduce a low-potential (higher energy) acceptor. This mechanism is now considered the third mode of energy transduction in biology, and offers promise for the development of novel bioinspired energy conversion strategies.

Bacteria "Read" Light To Gain a Competitive Advantage.

Photosynthesis, the process of converting solar energy into stored chemical bonds, represents the primary mechanism by which biological organisms utilize photons. Light can also be used to activate a number of photosensory compounds and proteins designed to carry out tasks, such as DNA repair, gene regulation, and synchronization with the diurnal cycle. Given that sunlight is incident upon many environments, it is not farfetched to think that life may have evolved other as-yet-undetected mechanisms to profit from solar irradiation.

The catalytic mechanism of electron-bifurcating electron transfer flavoproteins (ETFs) involves an intermediary complex with NAD.

Electron bifurcation plays a key role in anaerobic energy metabolism, but it is a relatively new discovery, and only limited mechanistic information is available on the diverse enzymes that employ it. Herein, we focused on the bifurcating electron transfer flavoprotein (ETF) from the hyperthermophilic archaeon The EtfABCX enzyme complex couples NADH oxidation to the endergonic reduction of ferredoxin and exergonic reduction of menaquinone. We developed a model for the enzyme structure by using nondenaturing MS, cross-linking, and homology modeling in which EtfA, -B, and -C each contained FAD, whereas EtfX contained two [4Fe-4S] clusters.

A new era for electron bifurcation.

Electron bifurcation, or the coupling of exergonic and endergonic oxidation-reduction reactions, was discovered by Peter Mitchell and provides an elegant mechanism to rationalize and understand the logic that underpins the Q cycle of the respiratory chain. Thought to be a unique reaction of respiratory complex III for nearly 40 years, about a decade ago Wolfgang Buckel and Rudolf Thauer discovered that flavin-based electron bifurcation is also an important component of anaerobic microbial metabolism. Their discovery spawned a surge of research activity, providing a basis to understand flavin-based bifurcation, forging fundamental parallels with Mitchell's Q cycle and leading to the proposal of metal-based bifurcating enzymes.

Distinct properties underlie flavin-based electron bifurcation in a novel electron transfer flavoprotein FixAB from .

A newly recognized third fundamental mechanism of energy conservation in biology, electron bifurcation, uses free energy from exergonic redox reactions to drive endergonic redox reactions. Flavin-based electron bifurcation furnishes low-potential electrons to demanding chemical reactions, such as reduction of dinitrogen to ammonia. We employed the heterodimeric flavoenzyme FixAB from the diazotrophic bacterium to elucidate unique properties that underpin flavin-based electron bifurcation.

Electron Bifurcation Makes the Puzzle Pieces Fall Energetically into Place in Methanogenic Energy Conservation.

Elaborate arrays of iron-sulfur clusters link active sites via a flavin that bifurcates electrons through two energetically independent paths. The structure of the heterodisulfide reductase provides insight into how methanogens conserve energy through coupling hydrogen oxidation to coordinated exergonic heterodisulfide and endergonic ferredoxin reduction in an overall thermodynamically favorable reaction.

Electron Bifurcation: Thermodynamics and Kinetics of Two-Electron Brokering in Biological Redox Chemistry.

How can proteins drive two electrons from a redox active donor onto two acceptors at very different potentials and distances? And how can this transaction be conducted without dissipating very much energy or violating the laws of thermodynamics? Nature appears to have addressed these challenges by coupling thermodynamically uphill and downhill electron transfer reactions, using two-electron donor cofactors that have very different potentials for the removal of the first and second electron. Although electron bifurcation is carried out with near perfection from the standpoint of energy conservation and electron delivery yields, it is a biological energy transduction paradigm that has only come into focus recently. This Account provides an exegesis of the biophysical principles that underpin electron bifurcation.

Activation Thermodynamics and H/D Kinetic Isotope Effect of the H to HH Transition in [FeFe] Hydrogenase.

Molecular complexes between CdSe nanocrystals and Clostridium acetobutylicum [FeFe] hydrogenase I (CaI) enabled light-driven control of electron transfer for spectroscopic detection of redox intermediates during catalytic proton reduction. Here we address the route of electron transfer from CdSe→CaI and activation thermodynamics of the initial step of proton reduction in CaI. The electron paramagnetic spectroscopy of illuminated CdSe:CaI showed how the CaI accessory FeS cluster chain (F-clusters) functions in electron transfer with CdSe.

The Electron Bifurcating FixABCX Protein Complex from Azotobacter vinelandii: Generation of Low-Potential Reducing Equivalents for Nitrogenase Catalysis.

The biological reduction of dinitrogen (N) to ammonia (NH) by nitrogenase is an energetically demanding reaction that requires low-potential electrons and ATP; however, pathways used to deliver the electrons from central metabolism to the reductants of nitrogenase, ferredoxin or flavodoxin, remain unknown for many diazotrophic microbes. The FixABCX protein complex has been proposed to reduce flavodoxin or ferredoxin using NADH as the electron donor in a process known as electron bifurcation. Herein, the FixABCX complex from Azotobacter vinelandii was purified and demonstrated to catalyze an electron bifurcation reaction: oxidation of NADH (E = -320 mV) coupled to reduction of flavodoxin semiquinone (E = -460 mV) and reduction of coenzyme Q (E = 10 mV).

Reduction Potentials of [FeFe]-Hydrogenase Accessory Iron-Sulfur Clusters Provide Insights into the Energetics of Proton Reduction Catalysis.

An [FeFe]-hydrogenase from Clostridium pasteurianum, CpI, is a model system for biological H activation. In addition to the catalytic H-cluster, CpI contains four accessory iron-sulfur [FeS] clusters in a branched series that transfer electrons to and from the active site. In this work, potentiometric titrations have been employed in combination with electron paramagnetic resonance (EPR) spectroscopy at defined electrochemical potentials to gain insights into the role of the accessory clusters in catalysis.