Tuning the redox properties of a [4Fe-4S] center to modulate the activity of Mo-bisPGD periplasmic nitrate reductase

Zeamari, Kamal; Gerbaud, Guillaume; Grosse, Sandrine; Fourmond, Vincent; Chaspoul, Florence; Biaso, Frédéric; Arnoux, Pascal; Sabaty, Monique; Pignol, David; Guigliarelli, Bruno; Burlat, Bénédicte

doi:10.1016/j.bbabio.2019.01.003

Cited by 13 publications

(11 citation statements)

References 59 publications

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“…Hence, the substoichiometric amount of W V species is likely not due to close values of E °(W VI/V ) and E °(W V/IV ) but more likely arises from the presence of different W species at the active site. Such substoichiometry and heterogeneity of the W V or Mo V EPR species are commonly observed in Mo/W-bisMGD enzymes. ,− In FdhAB, the strong increase in the W V species observed after enzyme activation and reduction by its substrate supports the likely involvement of these species in the catalytic mechanism.…”

Section: Results and Discussionmentioning

confidence: 68%

Spectroscopic and Structural Characterization of Reduced Desulfovibrio vulgaris Hildenborough W-FdhAB Reveals Stable Metal Coordination during Catalysis

et al. 2022

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Metal-dependent formate dehydrogenases are important enzymes due to their activity of CO 2 reduction to formate. The tungstencontaining FdhAB formate dehydrogenase from Desulfovibrio vulgaris Hildenborough is a good example displaying high activity, simple composition, and a notable structural and catalytic robustness. Here, we report the first spectroscopic redox characterization of FdhAB metal centers by EPR. Titration with dithionite or formate leads to reduction of three [4Fe−4S] 1+ clusters, and full reduction requires Ti(III)−citrate. The redox potentials of the four [4Fe−4S] 1+ centers range between −250 and −530 mV. Two distinct W V signals were detected, W D V and W F V, which differ in only the g 2 -value. This difference can be explained by small variations in the twist angle of the two pyranopterins, as determined through DFT calculations of model compounds. The redox potential of W VI/V was determined to be −370 mV when reduced by dithionite and −340 mV when reduced by formate. The crystal structure of dithionitereduced FdhAB was determined at high resolution (1.5 Å), revealing the same structural alterations as reported for the formatereduced structure. These results corroborate a stable six-ligand W coordination in the catalytic intermediate W V state of FdhAB.

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Section: Results and Discussionmentioning

confidence: 68%

Spectroscopic and Structural Characterization of Reduced Desulfovibrio vulgaris Hildenborough W-FdhAB Reveals Stable Metal Coordination during Catalysis

et al. 2022

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“…The typical Mo(V) EPR signatures detected in these samples are shown in Figure , while their redox behavior is depicted in Figure S5. The WT exhibits the two well characterized high pH and low pH Mo(V) species, both representing a maximal total amount of about 0.4 spin/NarGH (Figure A,B) which is typical of NarGH preparations. ,− More generally, such substoichiometric amounts of Mo(V) EPR species are commonly observed in the enzymes of the Mo- bis PGD family and are even lower in enzymes such as Nap, where they account for less than 0.2 spin/molecule. − In contrast, only the high pH Mo(V) species is detected in the E581Q variant with a maximal amount of 0.25 spin/NarGH (Figure C). This holds also true when the sample is prepared at pH 6.4 (Figure S6C), a value at which the low pH species is predominant in the WT (Figure S6A,B).…”

Section: Resultsmentioning

confidence: 97%

“…4A-B) which is typical of NarGH preparations (17,(19)(20)(21). More generally, such sub-stoichiometric amounts of Mo(V) EPR species are commonly observed in the enzymes of the Mo-bisPGD family and are even lower in enzymes such as Nap, where they account for less than 0.2 spin/molecule (72)(73)(74). In contrast, only the high pH Mo(V) species is detected in the E581Q variant with a maximal amount of 0.25 spin/NarGH (Fig.…”

Section: Influence Of the Hydrogen Bond Network Involving Glu-581 On ...mentioning

confidence: 97%

Gating of Substrate Access and Long-Range Proton Transfer in Escherichia coli Nitrate Reductase A: The Essential Role of a Remote Glutamate Residue

et al. 2021

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The Mo/W-bisPGD enzyme superfamily comprises a vast number of mononuclear molybdenum and tungsten enzymes that catalyze a great diversity of vital reactions in prokaryotes. In the past decades, much attention has been devoted to the immediate surroundings of the metal atom highlighting the importance of the inner coordination sphere but have failed to identify molecular determinants of the reactivity. Here, we report the mechanistic importance of a set of conserved residues that line the substrate entry tunnel in Escherichia coli nitrate reductase A (Nar), a paradigmatic enzyme of the Mo/W-bisPGD superfamily. Using mutagenesis, enzyme kinetics, electron paramagnetic resonance spectroscopy and molecular dynamics, we unveil the pivotal role of Glu-581 motion and a number of polar residues in its close proximity in substrate affinity and proton transfer to the Mo active site. Motion of the side chain of Glu-581 exhibiting a strong acid-base cooperativity with Asp-801 and surrounded by several polar interactions controls the hydration inside the protein core, proton transfer and substrate selectivity towards the active site. Overall, we identify an additional determinant that fine-tunes the reactivity and selectivity in Nar and propose that a gating mechanism is at play in several other members of the superfamily.

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“…Moreover, cluster I in mPCE2 becomes more temperature-sensitive and accounts for the overall broadening of EPR spectra only at a lower temperature. The increased line-width usually implies a significant effect of g -strain arising from an increased orientation distribution for a specific paramagnetic center upon freezing. − We thus speculated that the C8G-Y30N mutations resulted in a more solvent-exposed and dynamic conformation in the Fe B cluster.…”

Section: Results and Discussionmentioning

confidence: 99%

Rational Design of a Miniature Photocatalytic CO₂-Reducing Enzyme

Kang

Xia

et al. 2021

ACS Catal.

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Photosystem I (PSI) is a very large membrane protein complex (∼1000 kDa) harboring P700*, the strongest reductant known in biological systems, which is responsible for driving NAD(P) + and ultimately for CO 2 reduction. Although PSI is one of the most important components in the photosynthesis machinery, it has remained difficult to enhance PSI functions through genetic engineering due to its enormous complexity. Inspired by PSI's ability to undergo multiple-step photo-induced electron hopping from P700* to iron−sulfur [Fe 4 S 4 ] clusters, we designed a 33 kDa miniature photocatalytic CO 2 -reducing enzyme (mPCE) harboring a chromophore (BpC) and two [Fe 4 S 4 ] clusters (Fe A /Fe B ). Through reduction potential fine-tuning, we optimized the multiple-step electron hopping from BpC to Fe A /Fe B , culminating in a CO 2 / HCOOH conversion quantum efficiency of 1.43%. As mPCE can be overexpressed with a high yield in Escherichia coli cells without requiring synthetic cofactors, further development along this route may result in rapid photo-enzyme quantum yield improvement and functional expansion through an efficient directed evolution process. KEYWORDS: photosystem I, rational design, photosensitizer protein, iron−sulfur (Fe 4 S 4 ) cluster, photocatalytic, CO 2 reductase

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Tuning the redox properties of a [4Fe-4S] center to modulate the activity of Mo-bisPGD periplasmic nitrate reductase

Cited by 13 publications

References 59 publications

Spectroscopic and Structural Characterization of Reduced Desulfovibrio vulgaris Hildenborough W-FdhAB Reveals Stable Metal Coordination during Catalysis

Spectroscopic and Structural Characterization of Reduced Desulfovibrio vulgaris Hildenborough W-FdhAB Reveals Stable Metal Coordination during Catalysis

Gating of Substrate Access and Long-Range Proton Transfer in Escherichia coli Nitrate Reductase A: The Essential Role of a Remote Glutamate Residue

Rational Design of a Miniature Photocatalytic CO₂-Reducing Enzyme

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Tuning the redox properties of a [4Fe-4S] center to modulate the activity of Mo-bisPGD periplasmic nitrate reductase

Cited by 13 publications

References 59 publications

Spectroscopic and Structural Characterization of Reduced Desulfovibrio vulgaris Hildenborough W-FdhAB Reveals Stable Metal Coordination during Catalysis

Spectroscopic and Structural Characterization of Reduced Desulfovibrio vulgaris Hildenborough W-FdhAB Reveals Stable Metal Coordination during Catalysis

Gating of Substrate Access and Long-Range Proton Transfer in Escherichia coli Nitrate Reductase A: The Essential Role of a Remote Glutamate Residue

Rational Design of a Miniature Photocatalytic CO2-Reducing Enzyme

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Rational Design of a Miniature Photocatalytic CO₂-Reducing Enzyme