4.8 Article

The crystalline state as a dynamic system: IR microspectroscopy under electrochemical control for a [NiFe] hydrogenase

Journal

CHEMICAL SCIENCE
Volume 12, Issue 39, Pages 12959-12970

Publisher

ROYAL SOC CHEMISTRY
DOI: 10.1039/d1sc01734a

Keywords

-

Funding

  1. Biotechnology and Biological Sciences Research Council (BBSRC) [BB/R018413/1]
  2. European Research Council [ERC-2018-CoG BiocatSusChem 819580]
  3. Engineering and Physical Sciences Research Council (EPSRC) IB Catalyst award [EP/N013514/1]
  4. European Union's Horizon 2020 research and innovation programme under the Marie Sklodowska-Curie grant [752305]
  5. EPSRC [EP/N509711/1]
  6. BBSRC [BB/M011224/1]
  7. Ministerio de Ciencia y Tecnologia
  8. Universidad de Costa Rica
  9. Lincoln College, Oxford
  10. BBSRC [BB/R018413/1] Funding Source: UKRI
  11. EPSRC [EP/N013514/1] Funding Source: UKRI
  12. Marie Curie Actions (MSCA) [752305] Funding Source: Marie Curie Actions (MSCA)

Ask authors/readers for more resources

Controlled formation of catalytically-relevant states within crystals of complex metalloenzymes is a significant challenge, but electrochemical control over single crystals can stabilize specific states for further study and extend mechanistic understanding of proton transfer.
Controlled formation of catalytically-relevant states within crystals of complex metalloenzymes represents a significant challenge to structure-function studies. Here we show how electrochemical control over single crystals of [NiFe] hydrogenase 1 (Hyd1) from Escherichia coli makes it possible to navigate through the full array of active site states previously observed in solution. Electrochemical control is combined with synchrotron infrared microspectroscopy, which enables us to measure high signal-to-noise IR spectra in situ from a small area of crystal. The output reports on active site speciation via the vibrational stretching band positions of the endogenous CO and CN- ligands at the hydrogenase active site. Variation of pH further demonstrates how equilibria between catalytically-relevant protonation states can be deliberately perturbed in the crystals, generating a map of electrochemical potential and pH conditions which lead to enrichment of specific states. Comparison of in crystallo redox titrations with measurements in solution or of electrode-immobilised Hyd1 confirms the integrity of the proton transfer and redox environment around the active site of the enzyme in crystals. Slowed proton-transfer equilibria in the hydrogenase in crystallo reveals transitions which are only usually observable by ultrafast methods in solution. This study therefore demonstrates the possibilities of electrochemical control over single metalloenzyme crystals in stabilising specific states for further study, and extends mechanistic understanding of proton transfer during the [NiFe] hydrogenase catalytic cycle.

Authors

I am an author on this paper
Click your name to claim this paper and add it to your profile.

Reviews

Primary Rating

4.8
Not enough ratings

Secondary Ratings

Novelty
-
Significance
-
Scientific rigor
-
Rate this paper

Recommended

No Data Available
No Data Available