Importance of the protein framework for catalytic activity of [FeFe]-hydrogenases

Philipp Knörzer, Alexey Silakov, Carina E. Foster, Fraser A. Armstrong, Wolfgang Lubitz, Thomas Happe

Research output: Contribution to journalArticle

86 Citations (Scopus)

Abstract

The active center (H-cluster) of [FeFe]-hydrogenases is embedded into a hydrophobic pocket within the protein. We analyzed several amino acids, located in the vicinity of this niche, by site-directed mutagenesis of the [FeFe]-hydrogenases from Clostridium pasteurianum (CpI) and Chlamydomonas reinhardtii (CrHydA1). These amino acids are highly conserved and predicted to be involved in H-cluster coordination. Characterization of two hydrogenase variants confirmed this hypothesis. The exchange of residues CrHydA1Met 415 and CrHydA1Lys 228 resulted in inactive proteins, which, according to EPR and FTIR analyses, contain no intact H-cluster. However, [FeFe]-hydrogenases in which CpIMet 353(CrHydA1Met 223) and CpICys 299 (CrHydA1Cys 169) were exchanged to leucine and serine, respectively, showed a structurally intact H-cluster with catalytic activity either absent (CpIC299S) or strongly diminished (CpIM353L). In the case of CrHydA1C169S, the H-cluster was trapped in an inactive state exhibiting g values and vibrational frequencies that resembled the H trans state of DdH from Desulfovibrio desulfuricans. This cysteine residue, interacting with the bridge head nitrogen of the di(methyl)amine ligand, seems therefore to represent an essential contribution of the immediate protein environment to the reaction mechanism. Exchanging methionine CpIM 353(CrHydA1M 223) to leucine led to a strong decrease in turnover without affecting the K m value of the electron donor. We suggest that this methionine constitutes a "fine-tuning" element of hydrogenase activity.

Original languageEnglish (US)
Pages (from-to)1489-1499
Number of pages11
JournalJournal of Biological Chemistry
Volume287
Issue number2
DOIs
StatePublished - Jan 6 2012

Fingerprint

Hydrogenase
Catalyst activity
Proteins
Leucine
Methionine
Desulfovibrio desulfuricans
Amino Acids
Chlamydomonas reinhardtii
Clostridium
Mutagenesis
Vibrational spectra
Fourier Transform Infrared Spectroscopy
Site-Directed Mutagenesis
Serine
Amines
Cysteine
Paramagnetic resonance
Nitrogen
Tuning
Head

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Cell Biology
  • Molecular Biology

Cite this

Knörzer, Philipp ; Silakov, Alexey ; Foster, Carina E. ; Armstrong, Fraser A. ; Lubitz, Wolfgang ; Happe, Thomas. / Importance of the protein framework for catalytic activity of [FeFe]-hydrogenases. In: Journal of Biological Chemistry. 2012 ; Vol. 287, No. 2. pp. 1489-1499.
@article{3d4f998e13414be1b03922c5de9ee0a5,
title = "Importance of the protein framework for catalytic activity of [FeFe]-hydrogenases",
abstract = "The active center (H-cluster) of [FeFe]-hydrogenases is embedded into a hydrophobic pocket within the protein. We analyzed several amino acids, located in the vicinity of this niche, by site-directed mutagenesis of the [FeFe]-hydrogenases from Clostridium pasteurianum (CpI) and Chlamydomonas reinhardtii (CrHydA1). These amino acids are highly conserved and predicted to be involved in H-cluster coordination. Characterization of two hydrogenase variants confirmed this hypothesis. The exchange of residues CrHydA1Met 415 and CrHydA1Lys 228 resulted in inactive proteins, which, according to EPR and FTIR analyses, contain no intact H-cluster. However, [FeFe]-hydrogenases in which CpIMet 353(CrHydA1Met 223) and CpICys 299 (CrHydA1Cys 169) were exchanged to leucine and serine, respectively, showed a structurally intact H-cluster with catalytic activity either absent (CpIC299S) or strongly diminished (CpIM353L). In the case of CrHydA1C169S, the H-cluster was trapped in an inactive state exhibiting g values and vibrational frequencies that resembled the H trans state of DdH from Desulfovibrio desulfuricans. This cysteine residue, interacting with the bridge head nitrogen of the di(methyl)amine ligand, seems therefore to represent an essential contribution of the immediate protein environment to the reaction mechanism. Exchanging methionine CpIM 353(CrHydA1M 223) to leucine led to a strong decrease in turnover without affecting the K m value of the electron donor. We suggest that this methionine constitutes a {"}fine-tuning{"} element of hydrogenase activity.",
author = "Philipp Kn{\"o}rzer and Alexey Silakov and Foster, {Carina E.} and Armstrong, {Fraser A.} and Wolfgang Lubitz and Thomas Happe",
year = "2012",
month = "1",
day = "6",
doi = "10.1074/jbc.M111.305797",
language = "English (US)",
volume = "287",
pages = "1489--1499",
journal = "Journal of Biological Chemistry",
issn = "0021-9258",
publisher = "American Society for Biochemistry and Molecular Biology Inc.",
number = "2",

}

Importance of the protein framework for catalytic activity of [FeFe]-hydrogenases. / Knörzer, Philipp; Silakov, Alexey; Foster, Carina E.; Armstrong, Fraser A.; Lubitz, Wolfgang; Happe, Thomas.

In: Journal of Biological Chemistry, Vol. 287, No. 2, 06.01.2012, p. 1489-1499.

Research output: Contribution to journalArticle

TY - JOUR

T1 - Importance of the protein framework for catalytic activity of [FeFe]-hydrogenases

AU - Knörzer, Philipp

AU - Silakov, Alexey

AU - Foster, Carina E.

AU - Armstrong, Fraser A.

AU - Lubitz, Wolfgang

AU - Happe, Thomas

PY - 2012/1/6

Y1 - 2012/1/6

N2 - The active center (H-cluster) of [FeFe]-hydrogenases is embedded into a hydrophobic pocket within the protein. We analyzed several amino acids, located in the vicinity of this niche, by site-directed mutagenesis of the [FeFe]-hydrogenases from Clostridium pasteurianum (CpI) and Chlamydomonas reinhardtii (CrHydA1). These amino acids are highly conserved and predicted to be involved in H-cluster coordination. Characterization of two hydrogenase variants confirmed this hypothesis. The exchange of residues CrHydA1Met 415 and CrHydA1Lys 228 resulted in inactive proteins, which, according to EPR and FTIR analyses, contain no intact H-cluster. However, [FeFe]-hydrogenases in which CpIMet 353(CrHydA1Met 223) and CpICys 299 (CrHydA1Cys 169) were exchanged to leucine and serine, respectively, showed a structurally intact H-cluster with catalytic activity either absent (CpIC299S) or strongly diminished (CpIM353L). In the case of CrHydA1C169S, the H-cluster was trapped in an inactive state exhibiting g values and vibrational frequencies that resembled the H trans state of DdH from Desulfovibrio desulfuricans. This cysteine residue, interacting with the bridge head nitrogen of the di(methyl)amine ligand, seems therefore to represent an essential contribution of the immediate protein environment to the reaction mechanism. Exchanging methionine CpIM 353(CrHydA1M 223) to leucine led to a strong decrease in turnover without affecting the K m value of the electron donor. We suggest that this methionine constitutes a "fine-tuning" element of hydrogenase activity.

AB - The active center (H-cluster) of [FeFe]-hydrogenases is embedded into a hydrophobic pocket within the protein. We analyzed several amino acids, located in the vicinity of this niche, by site-directed mutagenesis of the [FeFe]-hydrogenases from Clostridium pasteurianum (CpI) and Chlamydomonas reinhardtii (CrHydA1). These amino acids are highly conserved and predicted to be involved in H-cluster coordination. Characterization of two hydrogenase variants confirmed this hypothesis. The exchange of residues CrHydA1Met 415 and CrHydA1Lys 228 resulted in inactive proteins, which, according to EPR and FTIR analyses, contain no intact H-cluster. However, [FeFe]-hydrogenases in which CpIMet 353(CrHydA1Met 223) and CpICys 299 (CrHydA1Cys 169) were exchanged to leucine and serine, respectively, showed a structurally intact H-cluster with catalytic activity either absent (CpIC299S) or strongly diminished (CpIM353L). In the case of CrHydA1C169S, the H-cluster was trapped in an inactive state exhibiting g values and vibrational frequencies that resembled the H trans state of DdH from Desulfovibrio desulfuricans. This cysteine residue, interacting with the bridge head nitrogen of the di(methyl)amine ligand, seems therefore to represent an essential contribution of the immediate protein environment to the reaction mechanism. Exchanging methionine CpIM 353(CrHydA1M 223) to leucine led to a strong decrease in turnover without affecting the K m value of the electron donor. We suggest that this methionine constitutes a "fine-tuning" element of hydrogenase activity.

UR - http://www.scopus.com/inward/record.url?scp=84855485266&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=84855485266&partnerID=8YFLogxK

U2 - 10.1074/jbc.M111.305797

DO - 10.1074/jbc.M111.305797

M3 - Article

VL - 287

SP - 1489

EP - 1499

JO - Journal of Biological Chemistry

JF - Journal of Biological Chemistry

SN - 0021-9258

IS - 2

ER -