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 i n the well-known EPR signals of [NiFe] hydrogenases. The preparation of the enzyme in several highly homogeneous states allowed us to carefully re-exa mine 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) li ne of the Ni-r* EPR signal. The hyperfine pattern became more pronounced in (H2O)-H-2. Simulations of the spectra suggested the interaction of the Ni- based unpaired electron with two equivalent, non-exchangeable protons (A(1. 2) = 13.2 MHz) and one exchangeable proton (A(3) = 6.6 MHz) in the Ni-r* st ate. Interaction with an exchangeable proton could not be observed in the N iu* EPR signal. The identity of the three protons is discussed and correlat ed 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.