Abstract
The selenium-containing F420-nonreducing hydrogenase from Methanococcus voltae was prepared in the Nia(I) middle dotCO state. The effect of illumination on this light-sensitive species was studied. EPR studies were carried out with enzyme containing natural selenium or with enzyme enriched in 77Se. Samples were prepared with either CO or 13CO. In the Nia(I) middle dotCO state, the nuclear spins of both 77Se (I = 1/2) and 13C (I = 1/2) interacted with the nickel-based unpaired electron, suggesting that they are positioned on opposite sites of the nickel ion. In the light-induced signal, the interaction with 13CO was lost. The 77Se nuclear spin introduced an anisotropic hyperfine splitting in both the dark and light-induced EPR signals. The data on the active enzyme of M. voltae are difficult to reconcile with the crystal structure of the inactive hydrogenase of Desulfovibrio gigas (Volbeda, A., Charon, M. H., Piras, C., Hatchikian, E. C., Frey, M., and Fontecilla Camps, J. C. (1995) Nature 373, 580-587) and suggest a structural change in the active site upon activation of the enzyme.
Highlights
Hydrogenases catalyze the heterolytic cleavage of molecular hydrogen into a hydride and a proton [1, 2], and the oxidation of the hydride to a proton and two electrons
The gz line of this signal is expected to be around g ϭ 2.02 [28, 29], but was hidden under additional lines originating from reduced Fe-S clusters and radical species in the sample
A comparison of traces A and B with trace D shows that the lines at g ϭ 2.21 and g ϭ 2.15 are due to the Nia(I)1⁄7H2 form and the line at g ϭ 2.28 and part of the mT; temperature, 35 K
Summary
We have reported [31] that the anaerobically purified selenium-containing F420-nonreducing hydrogenase from M. voltae displays a light-sensitive EPR signal with all the characteristics of the Nia(I)1⁄7H2 state (unpaired electron in an orbital with a predominantly dz character). No effects on the line widths could be observed when experiments were performed in D2O This means that in the Nia(I)1⁄7H2 state the unpaired spin in a dz orbital is pointing to Se, but not to hydrogen. Since it is assumed that CO and hydrogen bind to the same site [30], such a proposal can be checked by studying the Nia(I)1⁄713CO state in the Se-enzyme In this contribution we describe the EPR properties of COtreated selenium-containing F420-nonreducing hydrogenase from M. voltae. The results are discussed in light of the recent finding that the active site of [NiFe] hydrogenases is a bimetallic complex containing iron and nickel [32, 33]
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