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Title: Single-Amino Acid Modifications Reveal Additional Controls on the Proton Pathway of [FeFe]-Hydrogenase

Abstract

The proton pathway of [FeFe]-hydrogenase is essential for enzymatic H2 production and oxidation and is composed of four residues and a modeled water molecule. Recently, a computational analysis of this pathway revealed that the solvent-exposed residue of the pathway (Glu282) could form hydrogen bonds to two residues outside of the pathway (Arg286 and Ser320), implicating that these residues could function in regulating proton transfer. Substituting Arg286 with leucine eliminates hydrogen bonding with Glu282 and results in a 2.5-fold enhancement in H2 production activity, suggesting that Arg286 serves an important role in controlling the rate of proton delivery. In contrast, substitution of Ser320 with alanine reduces the rate approximately 5-fold, implying that it either acts as a member of the pathway or influences Glu282 to enable proton transfer. Interestingly, QM/MM and molecular dynamics calculations indicate that Ser320 does not play an electronic or structural role. QM calculations also estimate that including Ser320 in the pathway does not significantly change the barrier to proton movement, providing further support for its role as a member of the proton pathway. While further studies are needed to quantify the role of Ser320, collectively, these data provide evidence that the enzyme scaffold plays a significant rolemore » in modulating the activity of the enzyme, demonstrating that the rate of intraprotein proton transfer can be accelerated, particularly in a non-biological context. This work was supported by the DOE Great Lakes Bioenergy Research Center (DOE BER Office of Science, DE-FC02-07ER64494). In addition, support from the DOE Office of Science Early Career Research Program through the Office of Basic Energy Sciences (WJS, BGP, SR) is gratefully acknowledged. Computational resources were provided at W. R. Wiley Environmental Molecular Science Laboratory (EMSL), a national scientific user facility sponsored by the Department of Energy’s Office of Biological and Environmental Research located at Pacific Northwest National Laboratory, and a portion of the research was performed using PNNL Institutional Computing at Pacific Northwest National Laboratory. Pacific Northwest National Laboratory is operated by Battelle for the U.S. Department of Energy.« less

Authors:
; ; ; ; ; ; ; ;
Publication Date:
Research Org.:
Pacific Northwest National Lab. (PNNL), Richland, WA (United States)
Sponsoring Org.:
USDOE Office of Science (SC), Biological and Environmental Research (BER) (SC-23); USDOE Office of Science (SC), Basic Energy Sciences (BES) (SC-22)
OSTI Identifier:
1347864
Report Number(s):
PNNL-SA-111944
Journal ID: ISSN 0006-2960; KC0302010
DOE Contract Number:
AC05-76RL01830
Resource Type:
Journal Article
Resource Relation:
Journal Name: Biochemistry; Journal Volume: 55; Journal Issue: 22
Country of Publication:
United States
Language:
English
Subject:
catalysis; hydrogenase; proton pathway; outer coordination sphere

Citation Formats

Cornish, Adam J., Ginovska, Bojana, Thelen, Adam, da Silva, Julio C. S., Soares, Thereza A., Raugei, Simone, Dupuis, Michel, Shaw, Wendy J., and Hegg, Eric L. Single-Amino Acid Modifications Reveal Additional Controls on the Proton Pathway of [FeFe]-Hydrogenase. United States: N. p., 2016. Web. doi:10.1021/acs.biochem.5b01044.
Cornish, Adam J., Ginovska, Bojana, Thelen, Adam, da Silva, Julio C. S., Soares, Thereza A., Raugei, Simone, Dupuis, Michel, Shaw, Wendy J., & Hegg, Eric L. Single-Amino Acid Modifications Reveal Additional Controls on the Proton Pathway of [FeFe]-Hydrogenase. United States. doi:10.1021/acs.biochem.5b01044.
Cornish, Adam J., Ginovska, Bojana, Thelen, Adam, da Silva, Julio C. S., Soares, Thereza A., Raugei, Simone, Dupuis, Michel, Shaw, Wendy J., and Hegg, Eric L. Tue . "Single-Amino Acid Modifications Reveal Additional Controls on the Proton Pathway of [FeFe]-Hydrogenase". United States. doi:10.1021/acs.biochem.5b01044.
@article{osti_1347864,
title = {Single-Amino Acid Modifications Reveal Additional Controls on the Proton Pathway of [FeFe]-Hydrogenase},
author = {Cornish, Adam J. and Ginovska, Bojana and Thelen, Adam and da Silva, Julio C. S. and Soares, Thereza A. and Raugei, Simone and Dupuis, Michel and Shaw, Wendy J. and Hegg, Eric L.},
abstractNote = {The proton pathway of [FeFe]-hydrogenase is essential for enzymatic H2 production and oxidation and is composed of four residues and a modeled water molecule. Recently, a computational analysis of this pathway revealed that the solvent-exposed residue of the pathway (Glu282) could form hydrogen bonds to two residues outside of the pathway (Arg286 and Ser320), implicating that these residues could function in regulating proton transfer. Substituting Arg286 with leucine eliminates hydrogen bonding with Glu282 and results in a 2.5-fold enhancement in H2 production activity, suggesting that Arg286 serves an important role in controlling the rate of proton delivery. In contrast, substitution of Ser320 with alanine reduces the rate approximately 5-fold, implying that it either acts as a member of the pathway or influences Glu282 to enable proton transfer. Interestingly, QM/MM and molecular dynamics calculations indicate that Ser320 does not play an electronic or structural role. QM calculations also estimate that including Ser320 in the pathway does not significantly change the barrier to proton movement, providing further support for its role as a member of the proton pathway. While further studies are needed to quantify the role of Ser320, collectively, these data provide evidence that the enzyme scaffold plays a significant role in modulating the activity of the enzyme, demonstrating that the rate of intraprotein proton transfer can be accelerated, particularly in a non-biological context. This work was supported by the DOE Great Lakes Bioenergy Research Center (DOE BER Office of Science, DE-FC02-07ER64494). In addition, support from the DOE Office of Science Early Career Research Program through the Office of Basic Energy Sciences (WJS, BGP, SR) is gratefully acknowledged. Computational resources were provided at W. R. Wiley Environmental Molecular Science Laboratory (EMSL), a national scientific user facility sponsored by the Department of Energy’s Office of Biological and Environmental Research located at Pacific Northwest National Laboratory, and a portion of the research was performed using PNNL Institutional Computing at Pacific Northwest National Laboratory. Pacific Northwest National Laboratory is operated by Battelle for the U.S. Department of Energy.},
doi = {10.1021/acs.biochem.5b01044},
journal = {Biochemistry},
number = 22,
volume = 55,
place = {United States},
year = {Tue Jun 07 00:00:00 EDT 2016},
month = {Tue Jun 07 00:00:00 EDT 2016}
}
  • Possible proton channels in Clostridium pasteurianum [FeFe]-hydrogenase were investigated with molecular dynamics simulations. This study was undertaken to discern proposed channels, compare their properties, evaluate the functional channel, and to provide insight into the features of an active proton channel. Our simulations suggest that protons are not transported through water wires. Instead, a five-residue motif (E282, S319, E279, HOH, C299) was found to be the likely channel, consistent with experimental observations. This channel connects the surface of the enzyme and the di-thiomethylamine bridge of the catalytic H-cluster, permitting the transport of protons. The channel was found to have a persistentmore » hydrogen bonded core (residues E279 to S319), with less persistent hydrogen bonds at the ends of the channel. The hydrogen bond occupancy in this network was found to be sensitive to the protonation state of the residues in the channel, with different protonation states enhancing or stabilizing hydrogen bonding in different regions of the network. Single site mutations to non-hydrogen bonding residues break the hydrogen bonding network at that residue, consistent with experimental observations showing catalyst inactivation. In many cases, these mutations alter the hydrogen bonding in other regions of the channel which may be equally important in catalytic failure. A correlation between the protein dynamics near the proton channel and the redox partner binding regions was also found as a function of protonation state. The likely mechanism of proton movement in [FeFe]-hydrogenases is discussed based on the structural analysis presented here. This work was funded by the DOE Office of Science Early Career Research Program through the Office of Basic Energy Sciences. Computational resources were provided at W. R. Wiley Environmental Molecular Science Laboratory (EMSL), a national scientific user facility sponsored by the Department of Energy’s Office of Biological and Environmental Research located at Pacific Northwest National Laboratory, and a portion of the research was performed using PNNL Institutional Computing at Pacific Northwest National Laboratory. Pacific Northwest National Laboratory is operated by Battelle for the U.S. Department of Energy.« less