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.
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.
Bleijlevens
B
B
Swammerdam Institute for Life Sciences, Biochemistry, University of Amsterdam, Plantage Muidergracht 12, 1018 TV Amsterdam, The Netherlands.
Faber
B W
BW
Albracht
S P
SP
D016880:Anisotropy; D001665:Binding Sites; D002844:Chromatium / Q000201:enzymology*; D003198:Computer Simulation; D003903:Deuterium; D004578:Electron Spin Resonance Spectroscopy; D006864:Hydrogenase / Q000737:chemistry*; D007501:Iron / Q000737:chemistry; D009532:Nickel / Q000737:chemistry; D010084:Oxidation-Reduction; D011522:Protons
D011522:Protons; D009532:Nickel; D003903:Deuterium; D007501:Iron; C043296:nickel-iron hydrogenase; D006864:Hydrogenase