Structural Analysis of a Nitrogenase Iron Protein from Methanosarcina acetivorans: Implications for CO2 Capture by a Surface-Exposed [Fe4S4] Cluster.

Nitrogenase iron (Fe) proteins reduce CO2 to CO and/or hydrocarbons under ambient conditions. Here, we report a 2.4-Å crystal structure of the Fe protein from Methanosarcina acetivorans (MaNifH), which is generated in the presence of a reductant, dithionite, and an alternative CO2 source, bicarbonate. Structural analysis of this methanogen Fe protein species suggests that CO2 is possibly captured in an unactivated, linear conformation near the [Fe4S4] cluster of MaNifH by a conserved arginine (Arg) pair in a concerted and, possibly, asymmetric manner. Density functional theory calculations and mutational analyses provide further support for the capture of CO2 on MaNifH while suggesting a possible role of Arg in the initial coordination of CO2 via hydrogen bonding and electrostatic interactions. These results provide a useful framework for further mechanistic investigations of CO2 activation by a surface-exposed [Fe4S4] cluster, which may facilitate future development of FeS catalysts for ambient conversion of CO2 into valuable chemical commodities.IMPORTANCE This work reports the crystal structure of a previously uncharacterized Fe protein from a methanogenic organism, which provides important insights into the structural properties of the less-characterized, yet highly interesting archaeal nitrogenase enzymes. Moreover, the structure-derived implications for CO2 capture by a surface-exposed [Fe4S4] cluster point to the possibility of developing novel strategies for CO2 sequestration while providing the initial insights into the unique mechanism of FeS-based CO2 activation

A role for [Fe4S4] clusters in tRNA recognition—a theoretical study

Over the past several years, structural studies have led to the unexpected discovery of iron–sulfur clusters in enzymes that are involved in DNA replication/repair and protein biosynthesis. Although these clusters are generally well-studied cofactors, their significance in the new contexts often remains elusive. One fascinating example is a tryptophanyl-tRNA synthetase from the thermophilic bacterium Thermotoga maritima, TmTrpRS, that has recently been structurally characterized. It represents an unprecedented connection among a primordial iron–sulfur cofactor, RNA and protein biosynthesis. Here, a possible role of the [Fe4S4] cluster in tRNA anticodon-loop recognition is investigated by means of density functional theory and comparison with the structure of a human tryptophanyl-tRNA synthetase/tRNA complex. It turns out that a cluster-coordinating cysteine residue, R224, and polar main chain atoms form a characteristic structural motif for recognizing a putative 5′ cytosine or 5′ 2-thiocytosine moiety in the anticodon loop of the tRNA molecule. This motif provides not only affinity but also specificity by creating a structural and energetical penalty for the binding of other bases, such as uracil.ISSN:1362-4962ISSN:0301-561

Transcription factor-based biosensors for screening and dynamic regulation

[EN] Advances in synthetic biology and genetic engineering are bringing into the spotlight a wide range of bio-based applications that demand better sensing and control of biological behaviours. Transcription factor (TF)-based biosensors are promising tools that can be used to detect several types of chemical compounds and elicit a response according to the desired application. However, the wider use of this type of device is still hindered by several challenges, which can be addressed by increasing the current metabolite-activated transcription factor knowledge base, developing better methods to identify new transcription factors, and improving the overall workflow for the design of novel biosensor circuits. These improvements are particularly important in the bioproduction field, where researchers need better biosensor-based approaches for screening production-strains and precise dynamic regulation strategies. In this work, we summarize what is currently known about transcription factor-based biosensors, discuss recent experimental and computational approaches targeted at their modification and improvement, and suggest possible future research directions based on two applications: bioproduction screening and dynamic regulation of genetic circuits.JT-L was supported by European Union Marie Sklodowska-Curie Action Individual Postdoctoral Fellowship (Grant agreement ID: 101062593). JT-L was supported by the Next-Generation EU (NGEU) fund through the Spanish Recovery, Transformation and Resilience Plan via a Margarita Salas personal grant from the Spanish Ministry of Universities (UNI/551/2021). MS was supported by the NextGeneration EU (NGEU) fund through the Spanish Recovery, Transformation and Resilience Plan via a María Zambrano personal grant from the Spanish Ministry of Universities (UNI/551/2021). PC acknowledges MCIN/AEI/10.13039/501100011033 funding through PID2020-117271RB-C2 (BIODYNAMICS). PC was supported by the Spanish Ministry of Universities (UNI/551/2021), grant number UP 2021-036 funded by European Union - Next-generation EU. This research received financial support from Generalitat Valenciana through grant CIAICO/2021/159 (SmartBioFab). PC acknowledges MCIN/AEI/10.13039/501100011033 and NextGenerationEU/PRTR funding through grant TED 2021-131049 B-I00 (BioEcoDBTL). PC acknowledges MCIN/AEI/10.13039/501100011033 funding through grant PID 2021-127888NA-I00 (COMPSYNBIO).Tellechea-Luzardo, J.; Stiebritz, MT.; Carbonell, P. (2023). Transcription factor-based biosensors for screening and dynamic regulation. Frontiers in Bioengineering and Biotechnology. 11. https://doi.org/10.3389/fbioe.2023.11187021

Electric-Field Effects on the [FeFe]-Hydrogenase Active Site

The effect of a homogeneous electric field—as exerted by the protein environment and by an electrode potential—on the reactivity of the active site of [FeFe] hydrogenases is unravelled by density functional theory calculations.ISSN:1359-7345ISSN:1364-548

Regioselectivity of H Cluster Oxidation

The H₂-evolving potential of [FeFe] hydrogenases is severely limited by the oxygen sensitivity of this class of enzymes. Recent experimental studies on hydrogenase from C. reinhardtii point to O₂-induced structural changes in the [Fe₄S₄] subsite of the H cluster. Here, we investigate the mechanistic basis of this observation by means of density functional theory. Unexpectedly, we find that the isolated H cluster shows a pathological catalytic activity for the formation of reactive oxygen species such as O₂^– and HO₂^–. After protonation of O₂^–, an OOH radical may coordinate to the Fe atoms of the cubane, whereas H₂O₂ specifically reacts with the S atoms of the cubane-coordinating cysteine residues. Both pathways are accompanied by significant structural distortions that compromise cluster integrity and thus catalytic activity. These results explain the experimental observation that O₂-induced inhibition is accompanied by distortions of the [Fe₄S₄] moiety and account for the irreversibility of this process

Activation Barriers of Oxygen Transformation at the Active Site of [FeFe] Hydrogenases

Oxygen activation at the active sites of [FeFe] hydrogenases has been proposed to be the initial step of irreversible oxygen-induced inhibition of these enzymes. On the basis of a first theoretical study into the thermodynamics of O₂ activation [<i>Inorg. Chem.</i> <b>2009</b>, 48, 7127] we here investigate the kinetics of possible reaction paths at the distal iron atom of the active site by means of density functional theory. A sequence of steps is proposed to either form a reactive oxygen species (ROS) or fully reduce O₂ to water. In this reaction cascade, two branching points are identified where water formation directly competes with harmful oxygen activation reactions. The latter are water formation by O–O bond cleavage of a hydrogen peroxide-bound intermediate competing with H₂O₂ dissociation and CO₂ formation by a putative iron-oxo species competing with protonation of the iron-oxo species to form a hydroxyo ligand. Furthermore, we show that proton transfer to activated oxygen is fast and that proton supply to the active site is vital to prevent ROS dissociation. If sufficiently many reduction equivalents are available, oxygen activation reactions are accelerated, and oxygen reduction to water becomes possible

Kinetic Consequences of Introducing a Proximal Selenocysteine Ligand into Cytochrome P450cam

The structural, electronic, and catalytic properties of cytochrome P450cam are subtly altered when the cysteine that coordinates to the heme iron is replaced with a selenocysteine. To map the effects of the sulfur-to-selenium substitution on the individual steps of the catalytic cycle, we conducted a comparative kinetic analysis of the selenoenzyme and its cysteine counterpart. Our results show that the more electron-donating selenolate ligand has only negligible effects on substrate, product, and oxygen binding, electron transfer, catalytic turnover, and coupling efficiency. Off-pathway reduction of oxygen to give superoxide is the only step significantly affected by the mutation. Incorporation of selenium accelerates this uncoupling reaction approximately 50-fold compared to sulfur, but because the second electron transfer step is much faster, the impact on overall catalytic turnover is minimal. Density functional theory calculations with pure and hybrid functionals suggest that superoxide formation is governed by a delicate interplay of spin distribution, spin state, and structural effects. In light of the remarkably similar electronic structures and energies calculated for the sulfur- and selenium-containing enzymes, the ability of the heavier atom to enhance the rate of spin crossover may account for the experimental observations. Because the selenoenzyme closely mimics wild-type P450cam, even at the level of individual steps in the reaction cycle, selenium represents a unique mechanistic probe for analyzing the role of the proximal ligand and spin crossovers in P450 chemistry