Solvent-slaved protein motions accompany proton coupled electron transfer reactions catalysed by copper nitrite reductase

Hedison, Tobias M.; Heyes, Derren J.; Shanmugam, Maheswaran; Iorgu, Andreea I.; Scrutton, Nigel S.

doi:10.1039/c9cc01026b

Cited by 13 publications

(20 citation statements)

References 27 publications

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“…This long hydrogen bond and the indirect connection between WatA and WatB indicate that PT from bulk water needs thermodynamic fluctuations to rearrange hydrogen bond networks. This is consistent with that protein flexibility coupled with solvent motions controls proton transfer, which precedes the ET reaction in CuNIR (50). A slightly low occupancy of Wat2 implies its flexibility, which may be important to bring proton to the T2Cu site via Asp CAT .…”

Section: Resultssupporting

confidence: 84%

“…The O γ atom of Thr137 was a hydrogen donor to Asp112, the entrance residue of the MPC, and a hydrogen acceptor from the O γ atom of Ser96 that is a hydrogen acceptor from the amide moiety of Gly136. The observed hydrogen bond network shows the connection between the T1Cu site and the MPC, which may be involved in PCET accelerated by solvent-slaved protein motions (50).…”

Section: Resultsmentioning

confidence: 92%

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High-resolution neutron crystallography visualizes an OH-bound resting state of a copper-containing nitrite reductase

Fukuda

Hirano

Kusaka

et al. 2020

Proc. Natl. Acad. Sci. U.S.A.

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Copper-containing nitrite reductases (CuNIRs) transform nitrite to gaseous nitric oxide, which is a key process in the global nitrogen cycle. The catalytic mechanism has been extensively studied to ultimately achieve rational control of this important geobiochemical reaction. However, accumulated structural biology data show discrepancies with spectroscopic and computational studies; hence, the reaction mechanism is still controversial. In particular, the details of the proton transfer involved in it are largely unknown. This situation arises from the failure of determining positions of hydrogen atoms and protons, which play essential roles at the catalytic site of CuNIRs, even with atomic resolution X-ray crystallography. Here, we determined the 1.50 Å resolution neutron structure of a CuNIR from Geobacillus thermodenitrificans (trimer molecular mass of ∼106 kDa) in its resting state at low pH. Our neutron structure reveals the protonation states of catalytic residues (deprotonated aspartate and protonated histidine), thus providing insights into the catalytic mechanism. We found that a hydroxide ion can exist as a ligand to the catalytic Cu atom in the resting state even at a low pH. This OH-bound Cu site is unexpected from previously given X-ray structures but consistent with a reaction intermediate suggested by computational chemistry. Furthermore, the hydrogen-deuterium exchange ratio in our neutron structure suggests that the intramolecular electron transfer pathway has a hydrogen-bond jump, which is proposed by quantum chemistry. Our study can seamlessly link the structural biology to the computational chemistry of CuNIRs, boosting our understanding of the enzymes at the atomic and electronic levels.

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Section: Resultssupporting

confidence: 84%

Section: Resultsmentioning

confidence: 92%

High-resolution neutron crystallography visualizes an OH-bound resting state of a copper-containing nitrite reductase

Fukuda

Hirano

Kusaka

et al. 2020

Proc. Natl. Acad. Sci. U.S.A.

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“…Our examinations revealed that the driving force of intra‐ET comes from the remote water‐mediated triple‐proton transfer along the both proton channels. These results are consistent with the recent proposal of PT along the long water hydrogen bond network coupled ET in CuNiRs by experimental methods [64,65] . In general, the special PT/ET reactions further confirm the diversity of biological proton‐coupled ET mechanisms [66–71] …”

Section: Resultssupporting

confidence: 90%

“…These results are consistent with the recent proposal of PT along the long water hydrogen bond network coupled ET in CuNiRs by experimental methods. [64,65] In general, the special PT/ET reactions further confirm the diversity of biological proton-coupled ET mechanisms. [66][67][68][69][70][71]…”

Section: Long-range Pt Along the Primary Proton Channelmentioning

confidence: 63%

Remote Water‐Mediated Proton Transfer Triggers Inter‐Cu Electron Transfer: Nitrite Reduction Activation in Copper‐Containing Nitrite Reductase

Qin

Chen

2021

ChemBioChem

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The copper-containing nitrite reductase (CuNiR) catalyzes the biological conversion of nitrite to nitric oxide; key long-range electron/proton transfers are involved in the catalysis. However, the details of the electron-/proton-transfer mechanism are still unknown. In particular, the driving force of the electron transfer from the type-1 copper (T1Cu) site to the type-2 copper (T2Cu) site is ambiguous. Here, we explored the two possible protontransfer channels, the high-pH proton channel and the primary proton channel, by using two-layered ONIOM calculations. Our calculation results reveal that the driving force for electron transfer from T1Cu to T2Cu comes from a remote water-mediated triple-proton-coupled electron-transfer mechanism. In the high-pH proton channel, the water-mediated triple-proton transfer occurs from Glu113 to an intermediate water molecule, whereas in the primary channel, the transfer is from Lys128 to His260. Subsequently, the two channels employ another two or three distinct proton-transfer steps to deliver the proton to the nitrite substrate at the T2Cu site. These findings explain the detailed proton-/electron-transfer mechanisms of copper-containing nitrite reductase and could extend our understanding of the diverse proton-coupled electron-transfer mechanisms in complicated proteins.

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“…analysis. EPR measurements were carried out using a Bruker ELEXSYS-500/580 Xband EPR spectrometer operating in both continuous-wave (cw) and pulsed modes, equipped with an Oxford variabletemperature unit and ESR900 cryostat with Super High-Q resonator as reported previously (63)(64). All EPR samples were prepared in the quartz capillary tubes (outer diameter; 4.0 mm, inner diameter 3.0 mm) and flash-frozen immediately and stored in liquid N2 until analysis.…”

Section: Electron Paramagnetic Resonance (Epr)mentioning

confidence: 99%

Structural basis of carnitine monooxygenase CntA substrate specificity, inhibition, and intersubunit electron transfer

Quareshy

Shanmugam

Townsend

et al. 2021

Journal of Biological Chemistry

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Microbial metabolism of carnitine to trimethylamine (TMA) in the gut can accelerate atherosclerosis and heart disease and these TMA-producing enzymes are therefore important drug targets. Here, we report the first structures of the carnitine oxygenase CntA, an enzyme of the Rieske oxygenase family. CntA exists in a head-to-tail a3 trimeric structure. The two functional domains (the Rieske and the catalytic mononuclear iron domains) are located > 40 Å apart in the same monomer but adjacent in two neighbouring monomers. Structural determination of CntA and subsequent electron paramagnetic resonance measurements uncover the molecular basis of the so-called bridging glutamate (E205) residue in inter-subunit electron transfer. The structures of the substrate-bound CntA help to define the substrate pocket. Importantly, a tyrosine residue (Y203) is essential for ligand recognition through a π-cation interaction with the quaternary ammonium group. This interaction between an aromatic residue and quaternary amine substrates allows us to delineate a subgroup of Rieske oxygenases (group V) from the prototype ring-hydroxylating Rieske oxygenases involved in bioremediation of aromatic pollutants in the environment. Furthermore, we report the discovery of the first known CntA inhibitors and solve the structure of CntA in complex with the inhibitor, demonstrating the pivotal role of Y203 through a π-π stacking interaction with the inhibitor. Our study provides the structural and molecular basis for future discovery of drugs targeting this TMA-producing enzyme in human gut.

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Solvent-slaved protein motions accompany proton coupled electron transfer reactions catalysed by copper nitrite reductase

Abstract: A novel approach to study PCET reactions illustrates the importance of solvent-slaved protein motions in copper nitrite reductase catalysis.

Cited by 13 publications

References 27 publications

High-resolution neutron crystallography visualizes an OH-bound resting state of a copper-containing nitrite reductase

High-resolution neutron crystallography visualizes an OH-bound resting state of a copper-containing nitrite reductase

Remote Water‐Mediated Proton Transfer Triggers Inter‐Cu Electron Transfer: Nitrite Reduction Activation in Copper‐Containing Nitrite Reductase

Structural basis of carnitine monooxygenase CntA substrate specificity, inhibition, and intersubunit electron transfer

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