Flavodiiron Proteins Research Articles

Elimination of the flavodiiron electron sink facilitates long-term H2 photoproduction in green algae

BackgroundThe development of renewable and sustainable biofuels to cover the future energy demand is one of the most challenging issues of our time. Biohydrogen, produced by photosynthetic microorganisms, has the potential to become a green biofuel and energy carrier for the future sustainable world, since it provides energy without CO2 emission. The recent development of two alternative protocols to induce hydrogen photoproduction in green algae enables the function of the O2-sensitive [FeFe]-hydrogenases, located at the acceptor side of photosystem I, to produce H2 for several days. These protocols prevent carbon fixation and redirect electrons toward H2 production. In the present work, we employed these protocols to a knockout Chlamydomonas reinhardtii mutant lacking flavodiiron proteins (FDPs), thus removing another possible electron competitor with H2 production.ResultsThe deletion of the FDP electron sink resulted in the enhancement of H2 photoproduction relative to wild-type C. reinhardtii. Additionally, the lack of FDPs leads to a more effective obstruction of carbon fixation even under elongated light pulses.ConclusionsWe demonstrated that the rather simple adjustment of cultivation conditions together with genetic manipulation of alternative electron pathways of photosynthesis results in efficient re-routing of electrons toward H2 photoproduction. Furthermore, the introduction of a short recovery phase by regular switching from H2 photoproduction to biomass accumulation phase allows to maintain cell fitness and use photosynthetic cells as long-term H2-producing biocatalysts.

Role and regulation of class-C flavodiiron proteins in photosynthetic organisms.

The regulation of photosynthesis is crucial to efficiently support the assimilation of carbon dioxide and to prevent photodamage. One key regulatory mechanism is the pseudo-cyclic electron flow (PCEF) mediated by class-C flavodiiron proteins (FLVs). These enzymes use electrons coming from Photosystem I (PSI) to reduce oxygen to water, preventing over-reduction in the acceptor side of PSI. FLVs are widely distributed among organisms performing oxygenic photosynthesis and they have been shown to be fundamental in many different conditions such as fluctuating light, sulfur deprivation and plant submersion. Moreover, since FLVs reduce oxygen they can help controlling the redox status of the cell and maintaining the microoxic environment essential for processes such as nitrogen fixation in cyanobacteria. Despite these important roles identified in various species, the genes encoding for FLV proteins have been lost in angiosperms where their activity could have been at least partially compensated by a more efficient cyclic electron flow (CEF). The present work reviews the information emerged on FLV function, analyzing recent structural data that suggest FLV could be regulated through a conformational change.

Survival strategies of Clostridium difficile to fluctuating concentrations of oxygen

Oxygen and reactive oxygen species (ROS) can react with multiple cellular components, leading to the inactivation of a plethora of metabolic pathways. Therefore, organismshave systems to sense and eliminate O2 and ROS, contributing to their survival in the adverse host environment. Clostridium difficileP28 is an anaerobic pathogenic bacterium that contains in its genome a flavodiiron protein (FDP) and a rubrerythrin (Rbr),that are putatively involved in the detoxification pathways used by this organism. Flavodiiron proteins are widespread in all life domains, with a crucial role in O2detoxification, through its reduction directly to water. FDPs are cytoplasmic enzymes with a minimal structural unit composed by two main domains, a metallo-β-lactamase domain, containing the catalytic diiron site, and a flavodoxin domain having a flavin mononucleotide. Rubrerythrins are generally considered to act as NADH-linked hydrogen peroxide reductases, thus eliminating this ROS, and are composed by two iron sites:a diiron center and a rubredoxin-like FeCys4 center. In this work with characterized biochemically, spectroscopically, structurally and kinetically the FDP and Rbr and their twoputative redox partners, a High Molecular Weight Rubredoxin (HRb) and a Rubredoxin (Rd). We confirmed theexistence of direct electron transfer betweenHRb, Rd and FDP and also between HRb, Rd and Rbr. In addition, we alsoestablished the reaction rates for the reduction, by FDP, of O2(0.43s-1) and H2O2 (0.06s-1) and for the reduction of H2O2by Rbr (1.53s-1). The enzymatic activity of Rbr towards O2 was also investigated. Romão, C.V., et al, 2016.JBIC., 21:39-52. Martins, M.C., et al., 2019. FRBM, in press.

The role of metalloenzymes for the survival of the anaerobe Clostridium difficile during infection

Clostridium difficile is the most prevalent pathogen among all healthcare-associated infections. This anaerobic bacterium can colonize the human gut, typically following agents that disrupt the normal gut microbiota, like antibiotics. In the gut, C. difficile is subjected to oxygen, which it has to eliminate for survival. Its genome encodes for two flavodiiron proteins, capable to reduceoxygen to water. Besides, some FDPs also reduce NO to N2O, an important feature as a resistance mechanism towards the human innate immune system[1,2]. Flavodiiron enzymes are constituted by a minimal core of two domains: a metallo-β-lactamase-like one, harboring the catalytic center, followed by a short-chain flavodoxin[1,2]. More complex FDPs exist, with multiple extra domains and redox centers[1,2]. C. difficile contains a ‘classical’ FDP, and a very complex one, with an extra short-type rubredoxin domain followed by an NADH:rubredoxin oxidoreductase-like one[3]. The biochemical, redox and spectroscopic studies demonstrated that this enzyme receives electrons directly from NADH, reducing its substrates, precluding the need for extra partners. This FDP is selective for O2 (16s-1), almost 10x higher than with NO. The reactivity towards hydrogen peroxide, as an H2O2 reductase with formation of water, with a non-negligible turnover (2s-1) is a novelty in the field of FDPs. Site directed mutants revealed that the rubredoxin-like center is essential for electron transfer from NADH to the catalytic center. [1] Martins et al, Free Rad. Biol. Med., in press, 2019. [2] Romão et al, J. Biol.Inorg. Chem., 21,39-52, 2016. [3] Folgosa et al, Sci.Rep., 8, 10164, 2018.

Flavodiiron proteins 1-to-4 function in versatile combinations in O2 photoreduction in cyanobacteria.

Flavodiiron proteins (FDPs) constitute a group of modular enzymes widespread in Bacteria, Archaea and Eukarya. Synechocystis sp. PCC 6803 has four FDPs (Flv1-4), which are essential for the photoprotection of photosynthesis. A direct comparison of light-induced O2 reduction (Mehler-like reaction) under high (3% CO2, HC) and low (air level CO2, LC) inorganic carbon conditions demonstrated that the Flv1/Flv3 heterodimer is solely responsible for an efficient steady-state O2 photoreduction under HC, with flv2 and flv4 expression strongly down-regulated. Conversely, under LC conditions, Flv1/Flv3 acts only as a transient electron sink, due to the competing withdrawal of electrons by the highly induced NDH-1 complex. Further, in vivo evidence is provided indicating that Flv2/Flv4 contributes to the Mehler-like reaction when naturally expressed under LC conditions, or, when artificially overexpressed under HC. The O2 photoreduction driven by Flv2/Flv4 occurs down-stream of PSI in a coordinated manner with Flv1/Flv3 and supports slow and steady-state O2 photoreduction.

Alternative outlets for sustaining photosynthetic electron transport during dark-to-light transitions

Environmental stresses dramatically impact the balance between the production of photosynthetically derived energetic electrons and Calvin-Benson-Bassham cycle (CBBC) activity; an imbalance promotes accumulation of reactive oxygen species and causes cell damage. Hence, photosynthetic organisms have developed several strategies to route electrons toward alternative outlets that allow for storage or harmless dissipation of their energy. In this work, we explore the activities of three essential outlets associated with Chlamydomonas reinhardtii photosynthetic electron transport: (i) reduction of O2 to H2O through flavodiiron proteins (FLVs) and (ii) plastid terminal oxidases (PTOX) and (iii) the synthesis of starch. Real-time measurements of O2 exchange have demonstrated that FLVs immediately engage during dark-to-light transitions, allowing electron transport when the CBBC is not fully activated. Under these conditions, we quantified maximal FLV activity and its overall capacity to direct photosynthetic electrons toward O2 reduction. However, when starch synthesis is compromised, a greater proportion of the electrons is directed toward O2 reduction through both the FLVs and PTOX, suggesting an important role for starch synthesis in priming/regulating CBBC and electron transport. Moreover, partitioning energized electrons between sustainable (starch; energetic electrons are recaptured) and nonsustainable (H2O; energetic electrons are not recaptured) outlets is part of the energy management strategy of photosynthetic organisms that allows them to cope with the fluctuating conditions encountered in nature. Finally, unmasking the repertoire and control of such energetic reactions offers new directions for rational redesign and optimization of photosynthesis to satisfy global demands for food and other resources.

Thylakoid Localized Type 2 NAD(P)H Dehydrogenase NdbA Optimizes Light-Activated Heterotrophic Growth of Synechocystis sp. PCC 6803.

NdbA, one of the three type 2 NAD(P)H dehydrogenases (NDH-2) in Synechocystis sp. PCC 6803 (hereafter Synechocystis) was here localized to the thylakoid membrane (TM), unique for the three NDH-2s, and investigated with respect to photosynthetic and cellular redox metabolism. For this purpose, a deletion mutant (ΔndbA) and a complementation strain overexpressing NdbA (ΔndbA::ndbA) were constructed. It is demonstrated that NdbA is expressed at very low level in the wild-type (WT) Synechocystis under photoautotrophic (PA) growth whilst substantially higher expression occurs under light-activated heterotrophic growth (LAHG). The absence of NdbA resulted in non-optimal growth of Synechocystis under LAHG and concomitantly enhanced the expression of photoprotection-related flavodiiron proteins and carbon acquisition-related proteins as well as various transporters, but downregulated a few iron homeostasis-related proteins. NdbA overexpression, on the other hand, promoted photosynthetic pigmentation and functionality of photosystem I under LAHG conditions while distinct photoprotective and carbon concentrating proteins were downregulated. NdbA overexpression also exerted an effect on the expression of many signaling and gene regulation proteins. It is concluded that the amount and function of NdbA in the TM has a capacity to modulate the redox signaling of gene expression, but apparently has a major physiological role in maintaining iron homeostasis under LAHG conditions. LC-MS/MS data are available via ProteomeXchange with identifier PXD011671.

The oxygen reduction reaction catalyzed by Synechocystis sp. PCC 6803 flavodiiron proteins

Photosynthetic flavodiiron proteins catalyze oxygen reduction at non-heme iron sites (brown spheres) using flavin (FMN) mediated electron transfer (black arrows).

Role of cyclic and pseudo-cyclic electron transport in response to dynamic light changes in Physcomitrella patens.

Photosynthetic organisms support cell metabolism by harvesting sunlight and driving the electron transport chain at the level of thylakoid membranes. Excitation energy and electron flow in the photosynthetic apparatus is continuously modulated in response to dynamic environmental conditions. Alternative electron flow around photosystem I plays a seminal role in this regulation contributing to photoprotection by mitigating overreduction of the electron carriers. Different pathways of alternative electron flow coexist in the moss Physcomitrella patens, including cyclic electron flow mediated by the PGRL1/PGR5 complex and pseudo-cyclic electron flow mediated by the flavodiiron proteins FLV. In this work, we generated P.patens plants carrying both pgrl1 and flva knock-out mutations. A comparative analysis of the WT, pgrl1, flva, and pgrl1 flva lines suggests that cyclic and pseudo-cyclic processes have a synergic role in the regulation of photosynthetic electron transport. However, although both contribute to photosystem I protection from overreduction by modulating electron flow following changes in environmental conditions, FLV activity is particularly relevant in the first seconds after a light change whereas PGRL1 has a major role upon sustained strong illumination.

Analysis of a new flavodiiron core structural arrangement in Flv1-ΔFlR protein from Synechocystis sp. PCC6803

Flavodiiron proteins (FDPs) play key roles in biological response mechanisms against oxygen and/or nitric oxide; in particular they are present in oxygenic phototrophs (including cyanobacteria and gymnosperms). Two conserved domains define the core of this family of proteins: a N-terminal metallo-β-lactamase-like domain followed by a C-terminal flavodoxin-like one, containing the catalytic diiron centre and a FMN cofactor, respectively. Members of the FDP family may present extra modules in the C-terminus, and were classified into several classes according to their distribution and composition. The cyanobacterium Synechocystis sp. PCC6803 contains four Class C FDPs (Flv1-4) that include at the C-terminus an additional NAD(P)H:flavin oxidoreductase (FlR) domain. Two of them (Flv3 and Flv4) have the canonical diiron ligands (Class C, Type 1), while the other two (Flv1 and Flv2) present different residues in that region (Class C, Type 2). Most phototrophs, either Bacterial or Eukaryal, contain at least two FDP genes, each encoding for one of those two types. Crystals of the Flv1 two core domains (Flv1-ΔFlR), without the C-terminal NAD(P)H:flavin oxidoreductase extension, were obtained and the structure was determined. Its pseudo diiron site contains non-canonical basic and neutral residues, and showed anion moieties, instead. The presented structure revealed for the first time the structure of the two-domain core of a Class C-Type 2 FDP.

Spectroscopy and DFT Calculations of Flavo-Diiron Nitric Oxide Reductase Identify Bridging Structures of NO-Coordinated Diiron Intermediates.

Flavo-diiron proteins (FDPs) are widespread in anaerobic bacteria, archaea, and protozoa, where they serve as the terminal components of dioxygen and nitric oxide reductive scavenging pathways. FDPs contain an N,O-ligated diiron site adjacent to a flavin mononucleotide (FMN) cofactor. The diiron site is structurally similar to those in hemerythrin, ribonucleotide reductase, and methane monooxygenase. However, only FDPs turn over NO to N2O at significant rates and yields. Previous studies revealed sequential binding of two NO molecules to the diferrous site, forming mono- and dinitrosyl intermediates leading to N2O formation. In the present work, these mono- and dinitrosyl intermediates have been characterized by EPR and Mössbauer spectroscopies and DFT calculations. Our results show that the iron proximal to the cofactor binds the first NO to form the diiron mononitrosyl complex, implying the iron distal to the FMN binds the second NO to form the diiron dinitrosyl intermediate. The exchange-coupling constants, J (H = JS1·S2), were found to differ substantially, +17 cm-1 for the diiron mononitrosyl and +60 cm-1 for the diiron dinitrosyl. Notwithstanding this large difference, our findings indicate retention of at least one hydroxo bridge throughout the NOR catalytic cycle. The Mossbauer hyperfine parameters and DFT calculations confirmed a semibridging NO- ligand in the mononitrosyl intermediate that lowers the exchange parameter. The DFT calculations on the dinitrosyl intermediate suggest a contribution to J from direct exchange between the S = 1 spins on the NO- ligands, which could initiate N-N bond formation. Our results provide insight into why FDPs are the only known nonheme diiron enzymes that competently turn over NO to N2O.

Balancing protection and efficiency in the regulation of photosynthetic electron transport across plant evolution

Contents Summary 105 I. Introduction 105 II. Diversity of molecular mechanisms for regulation of photosynthetic electron transport 106 III. Role of FLVs in the regulation of photosynthesis in eukaryotes 107 IV. Why were FLVs lost in angiosperms? 108 V. Conclusions 108 Acknowledgements 109 References 109 SUMMARY: Photosynthetic electron transport requires continuous modulation to maintain the balance between light availability and metabolic demands. Multiple mechanisms for the regulation of electron transport have been identified and are unevenly distributed among photosynthetic organisms. Flavodiiron proteins (FLVs) influence photosynthetic electron transport by accepting electrons downstream of photosystemI to reduce oxygen to water. FLV activity has been demonstrated in cyanobacteria, green algae and mosses to be important in avoiding photosystemI overreduction upon changes in light intensity. FLV-encoding sequences were nevertheless lost during evolution by angiosperms, suggesting that these plants increased the efficiency of other mechanisms capable of accepting electrons from photosystemI, making the FLV activity for protection from overreduction superfluous or even detrimental for photosynthetic efficiency.

The multidomain flavodiiron protein from Clostridium difficile 630 is an NADH:oxygen oxidoreductase

Flavodiiron proteins (FDPs) are enzymes with a minimal core of two domains: a metallo-β-lactamase-like, harbouring a diiron center, and a flavodoxin, FMN containing, domains. FDPs are O2 or NO reducing enzymes; for many pathogens, they help mitigate the NO produced by the immune system of the host, and aid survival during fluctuating concentrations concentrations of oxygen. FDPs have a mosaic structure, being predicted to contain multiple extra domains. Clostridium difficile, a threatening human pathogen, encodes two FDPs: one with the two canonical domains, and another with a larger polypeptide chain of 843 amino acids, CD1623, with two extra domains, predicted to be a short-rubredoxin-like and an NAD(P)H:rubredoxin oxidoreductase. This multi-domain protein is the most complex FDP characterized thus far. Each of the predicted domains was characterized and the presence of the predicted cofactors confirmed by biochemical and spectroscopic analysis. Results show that this protein operates as a standalone FDP, receiving electrons directly from NADH, and reducing oxygen to water, precluding the need for extra partners. CD1623 displayed negligible NO reductase activity, and is thus considered an oxygen selective FDP, that may contribute to the survival of C. difficile in the human gut and in the environment.

Flavodiiron-Mediated O2 Photoreduction Links H2 Production with CO2 Fixation during the Anaerobic Induction of Photosynthesis.

Some microalgae, such as Chlamydomonas reinhardtii, harbor a highly flexible photosynthetic apparatus capable of using different electron acceptors, including carbon dioxide (CO2), protons, or oxygen (O2), allowing survival in diverse habitats. During anaerobic induction of photosynthesis, molecular O2 is produced at photosystem II, while at the photosystem I acceptor side, the reduction of protons into hydrogen (H2) by the plastidial [FeFe]-hydrogenases primes CO2 fixation. Although the interaction between H2 production and CO2 fixation has been studied extensively, their interplay with O2 produced by photosynthesis has not been considered. By simultaneously measuring gas exchange and chlorophyll fluorescence, we identified an O2 photoreduction mechanism that functions during anaerobic dark-to-light transitions and demonstrate that flavodiiron proteins (Flvs) are the major players involved in light-dependent O2 uptake. We further show that Flv-mediated O2 uptake is critical for the rapid induction of CO2 fixation but is not involved in the creation of the micro-oxic niches proposed previously to protect the [FeFe]-hydrogenase from O2 By studying a mutant lacking both hydrogenases (HYDA1 and HYDA2) and both Flvs (FLVA and FLVB), we show that the induction of photosynthesis is strongly delayed in the absence of both sets of proteins. Based on these data, we propose that Flvs are involved in an important intracellular O2 recycling process, which acts as a relay between H2 production and CO2 fixation.

Photoinduction of electron transport on the acceptor side of PSI in Synechocystis PCC 6803 mutant deficient in flavodiiron proteins Flv1 and Flv3

After transferring the dark-acclimated cyanobacteria to light, flavodiiron proteins Flv1/Flv3 serve as a main electron acceptor for PSI within the first seconds because Calvin cycle enzymes are inactive in the dark. Synechocystis PCC 6803 mutant Δflv1/Δflv3 devoid of Flv1 and Flv3 retained the PSI chlorophyll P700 in the reduced state over 10 s (Helman et al., 2003; Allahverdiyeva et al., 2013). Study of P700 oxidoreduction transients in dark-acclimated Δflv1/Δflv3 mutant under the action of successive white light pulses separated by dark intervals of various durations indicated that the delayed oxidation of P700 was determined by light activation of electron transport on the acceptor side of PSI. We show that the light-induced redox transients of chlorophyll P700 in dark-acclimated Δflv1/Δflv3 proceed within 2 min, as opposed to 1–3 s in the wild type, and comprise a series of kinetic stages. The release of rate-limiting steps was eliminated by iodoacetamide, an inhibitor of Calvin cycle enzymes. Conversely, the creation with methyl viologen of a bypass electron flow to O2 accelerated P700 oxidation and made its extent comparable to that in the wild-type cells. The lack of major sinks for linear electron flow in iodoacetamide-treated Δflv1/Δflv3 mutant, in which O2- and CO2-dependent electron flows were impaired, facilitated cyclic electron flow, which was evident from the decreased steady-state oxidation of P700 and from rapid dark reduction of P700 during and after illumination with far-red light. The results show that the photosynthetic induction in wild-type Synechocystis PCC 6803 is largely hidden due to the flavodiiron proteins whose operation circumvents the rate-limiting electron transport steps controlled by Calvin cycle reactions.

Comparative analysis of strategies to prepare electron sinks in aquatic photoautotrophs.

While subject to illumination, photosystem I (PSI) has the potential to produce reactive oxygen species (ROS) that can cause photo-oxidative damage in oxygenic photoautotrophs. The reaction center chlorophyll in PSI (P700) is kept oxidized in excess light conditions to limit over-excitation of PSI and alleviate the production of ROS. Oxidation of P700 requires a sufficient electron sink for PSI, which is responsible for flavodiiron proteins (FLV) safely dissipating electrons to O2 in cyanobacteria, green algae, and land plants except for angiosperms during short-pulse light (SP) illumination under which photosynthesis and photorespiration do not occur. This fact implies that O2 usage is essential for P700 oxidation but also raises the question why angiosperms lost FLV. Here, we first found that aquatic photoautotrophs in red plastid lineage, in which no gene for FLV has been found, could keep P700 oxidized during SP illumination alleviating the photo-oxidative damage in PSI even without O2 usage. We comprehensively assessed P700 oxidation during SP illumination in the presence and absence of O2 in cyanobacteria (Cyanophyta), green algae (Chlorophyta), angiosperms (Streptophyta), red algae (Rhodophyta), and secondary algae (Cryptophyta, Haptophyta, and Heterokontophyta). A variety of dependencies of P700 oxidation on O2 among these photoautotrophs clearly suggest that O2 usage and FLV are not universally required to oxidize P700 for protecting PSI against ROS damage. Our results expand the understanding of the diverse strategies taken by oxygenic photoautotrophs to oxidize P700 and mitigate the risks of ROS.

P-67 - Diverse strategies in P700 oxidation system for suppressing ROS production were acquired during the evolution of photosynthetic organisms

We developed the method to produce ROS intrinsically in photosynthetic organisms: repetitive short-pulse (rSP) illumination. rSP illumination fulfills electrons in photosynthetic electron transport (PET) system, where O2 is reduced to superoxide radical and O2 is also excited to singlet O2 at photosystem I (PSI) of PET system. These ROS oxidatively damage PSI in angiosperms. By contrast, mosses/liverworts, ferns, and gymnosperms do not suffer from PSI damages, where a reaction center chlorophyll in PSI, P700, was oxidized to the maximum in the presence of O2, on the other hand P700 was reduced in angiosperm leaves. The reduced P700 shows electron accumulation in PSI to stimulate ROS production. These organisms, other than angiosperms, have flavodiiron (FLV) proteins, which catalyze O2 reduction to H2O using ferredoxin/NADPH. The mutant of liverwort, Marchantia polymorpha, deficient in FLV, could not oxidize P700 and suffered from PSI oxidative damage by rSP-illumination. That is, these photosynthetic organisms rapidly oxidize P700 to suppress ROS production. Recently, we found that diatoms, red algae, brown algae oxidize P700 even in the absence of O2 and did not suffer from any oxidative damages of PSI. These photosynthetic organisms might have a new mechanism to oxidize P700.

Hunting the main player enabling Chlamydomonas reinhardtii growth under fluctuating light.

Photosynthetic organisms have evolved numerous photoprotective mechanisms and alternative electron sinks/pathways to fine-tune the photosynthetic apparatus under dynamic environmental conditions, such as varying carbon supply or fluctuations in light intensity. In cyanobacteria flavodiiron proteins (FDPs) protect the photosynthetic apparatus from photodamage under fluctuating light (FL). In Arabidopsis thaliana, which does not possess FDPs, the PGR5-related pathway enables FL photoprotection. The direct comparison of the pgr5, pgrl1 and flv knockout mutants of Chlamydomonas reinhardtii grown under ambient air demonstrates that all three proteins contribute to the survival of cells under FL, but to varying extents. The FDPs are crucial in providing a rapid electron sink, with flv mutant lines unable to survive even mild FL conditions. In contrast, the PGRL1 and PGR5-related pathways operate over relatively slower and longer time-scales. Whilst deletion of PGR5 inhibits growth under mild FL, the pgrl1 mutant line is only impacted under severe FL conditions. This suggests distinct roles, yet a close relationship, between the function of PGR5, PGRL1 and FDP proteins in photoprotection.

The Semireduced Mechanism for Nitric Oxide Reduction by Non-Heme Diiron Complexes: Modeling Flavodiiron Nitric Oxide Reductases.

Flavodiiron nitric oxide reductases (FNORs) are a subclass of flavodiiron proteins (FDPs) capable of preferential binding and subsequent reduction of NO to N2O. FNORs are found in certain pathogenic bacteria, equipping them with resistance to nitrosative stress, generated as a part of the immune defense in humans, and allowing them to proliferate. Here, we report the spectroscopic characterization and detailed reactivity studies of the diiron dinitrosyl model complex [Fe2(BPMP)(OPr)(NO)2](OTf)2 for the FNOR active site that is capable of reducing NO to N2O [Zheng et al., J. Am. Chem. Soc. 2013, 135, 4902-4905]. Using UV-vis spectroscopy, cyclic voltammetry, and spectro-electrochemistry, we show that one reductive equivalent is in fact sufficient for the quantitative generation of N2O, following a semireduced reaction mechanism. This reaction is very efficient and produces N2O with a first-order rate constant k > 102 s-1. Further isotope labeling studies confirm an intramolecular N-N coupling mechanism, consistent with the rapid time scale of the reduction and a very low barrier for N-N bond formation. Accordingly, the reaction proceeds at -80 °C, allowing for the direct observation of the mixed-valent product of the reaction. At higher temperatures, the initial reaction product is unstable and decays, ultimately generating the diferrous complex [Fe2(BPMP)(OPr)2](OTf) and an unidentified ferric product. These results combined offer deep insight into the mechanism of NO reduction by the relevant model complex [Fe2(BPMP)(OPr)(NO)2]2+ and provide direct evidence that the semireduced mechanism would constitute a highly efficient pathway to accomplish NO reduction to N2O in FNORs and in synthetic catalysts.

Diversity and complexity of flavodiiron NO/O2 reductases.

Flavodiiron proteins (FDPs) are a family of enzymes endowed with nitric oxide (NO) or oxygen reductase activities, forming the innocuous nitrous oxide (N2O) or water molecules, respectively. FDPs are widespread in the three life kingdoms, and have a modular nature, being each monomer minimally constituted by a metallo-β-lactamase-like domain containing a catalytic diiron centre, followed by a flavodoxin one, with a flavin mononucleotide. Since their discovery, additional domains have been found in FDPs, attached to the C-terminus, and containing either extra metal (iron) centers or extra flavin binding modules. Following an extensive analysis of genomic databases, we identified novel domain compositions, and proposed a new classification of FDPs in eight classes based on the nature and number of extra domains.