Enzyme electrokinetics: Electrochemical studies of the anaerobic interconversions between active and inactive states of Allochromatium vinosum [NiFe]-hydrogenase

Anne K. Jones, Sophie E. Lamle, Harsh R. Pershad, Kylie A. Vincent, Simon P.J. Albracht, Fraser A. Armstrong

Research output: Contribution to journalArticlepeer-review

150 Scopus citations


The cycling between active and inactive states of the catalytic center of [NiFe]-hydrogenase from Allochromatium vinosum has been investigated by dynamic electrochemical techniques. Adsorbed on a rotating disk pyrolytic graphite "edge" electrode, the enzyme is highly electroactive: this allows precise manipulations of the complex redox chemistry and facilitates quantitative measurements of the interconversions between active catalytic states and the inactive oxidized form Nir* (also called Ni-B or "ready") as functions of pH, H2 partial pressure, temperature, and electrode potential. Cyclic voltammograms for catalytic H2 oxidation (current is directly related to turnover rate) are highly asymmetric (except at pH > 8 and high temperature) due to inactivation being much slower than activation. Controlled potential-step experiments show that the rate of oxidative inactivation increases at high pH but is independent of potential, whereas the rate of reductive activation increases as the potential becomes more negative. Indeed, at 45 °C, activation takes just a few seconds at -288 mV. The cyclic asymmetry arises because interconversion is a two-stage reaction, as expected if the reduced inactive Nir-S state is an intermediate. The rate of inactivation depends on a chemical process (rearrangement and uptake of a ligand) that is independent of potential, but sensitive to pH, while activation is driven by an electron-transfer process, Ni(III) to Ni(II), that responds directly to the driving force. The potentials at which fast activation occurs under different conditions have been analyzed to yield the potential-pH dependence and the corresponding entropies and enthalpies. The reduced (active) enzyme shows a pK of 7.6; thus, when a one-electron process is assumed, reductive activation at pH < 7 involves a net uptake of one proton (or release of one hydroxide), whereas, at pH > 8, there is no net exchange of protons with solvent. Activation is favored by a large positive entropy, consistent with the release of a ligand and/or relaxation of the structure around the active site.

Original languageEnglish (US)
Pages (from-to)8505-8514
Number of pages10
JournalJournal of the American Chemical Society
Issue number28
StatePublished - Jul 16 2003
Externally publishedYes

ASJC Scopus subject areas

  • Catalysis
  • Chemistry(all)
  • Biochemistry
  • Colloid and Surface Chemistry


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