The [NiFe] hydrogenase from Allochromatium vinosum studied in EPR-detectable states: H/D exchange experiments that yield new information about the structure of the active site

• Published in: Journal of biological inorganic chemistry 2001-10, Vol.6 (8), p.763-769
• Main Authors: Bleijlevens, B, Faber, B W, Albracht, S P
• Format: Article
• Language: English
• Subjects: Anisotropy; Binding Sites; Chromatium - enzymology; Computer Simulation; Deuterium; Electron Spin Resonance Spectroscopy; Hydrogenase - chemistry; Iron - chemistry; Nickel - chemistry; Oxidation-Reduction; Protons
• ISSN: 0949-8257; 1432-1327
• DOI: 10.1007/s007750100252

In this study we report on thus-far unobserved proton hyperfine couplings in the well-known EPR signals of [NiFe] hydrogenases. The preparation of the enzyme in several highly homogeneous states allowed us to carefully re-examine the Ni(u)*, Ni(r)*, Ni(a)-C* and Ni(a)-L* EPR signals which are present in most [NiFe] hydrogenases. At high resolution (modulation amplitude 0.57 G), clear indications for hyperfine interactions were observed in the g(z) line of the Ni(r)* EPR signal. The hyperfine pattern became more pronounced in 2H2O. Simulations of the spectra suggested the interaction of the Ni-based unpaired electron with two equivalent, non-exchangeable protons (A1,2=13.2 MHz) and one exchangeable proton (A3=6.6 MHz) in the Ni(r)* state. Interaction with an exchangeable proton could not be observed in the Ni(u)* EPR signal. The identity of the three protons is discussed and correlated to available ENDOR data. It is concluded that the NiFe centre in the Ni(r)* state contains a hydroxide ligand bound to the nickel, which is pointing towards the gas channel rather than to iron.