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Hydrogenases are the most active molecular catalysts for hydrogen production and uptake (1,2), and could therefore facilitate the development of new types of fuel cell (3-5). In [FeFe]-hydrogenases, catalysis takes place at a unique di-iron centre (the [2Fe] subsite), which contains a bridging dithiolate ligand, three CO ligands and two [CN.sup.-] ligands (6,7). Through a complex multienzymatic biosynthetic process, this [2Fe] subsite is first assembled on a maturation enzyme, HydF, and then delivered to the apo-hydrogenase for activation (8). Synthetic chemistry has been used to prepare remarkably similar mimics of that subsite (1), but it has failed to reproduce the natural enzymatic activities thus far. Here we show that three synthetic mimics (containing different bridging dithiolate ligands) can be loaded onto bacterial Thermotoga maritima HydF and then transferred to apo-HydA1, one of the hydrogenases of Chlamydomonas reinhardtii algae. Full activation of HydAl was achieved only when using the HydF hybrid protein containing the mimic with an azadithiolate bridge, confirming the presence of this ligand in the active site of native [FeFe]-hydrogenases (9,10). This is an example of controlled metalloenzyme activation using the combination of a specific protein scaffold and active-site synthetic analogues. This simple methodology provides both new mechanistic and structural insight into hydrogenase maturation and a unique tool for producing recombinant wild-type and variant [FeFe]-hydrogenases, with no requirement for the complete maturation machinery., Complexes 1 (11-13), 2 (14) and 3 (15) (Fig. 1a) represent the closest synthetic mimics of the [2Fe] subsite in HydA1. They all share the same primary coordination sphere with [...]

'Site values' are used to obtain a set of spin projection coefficients for different iron sulfur clusters in different oxidation states. The best spin state in a pairwise model having two ions dimer combined to a tetramer is (7/2,3,1/2), for the (Fe4S4)3+ cluster. For 4Fe ferrodoxins, the spin projection coefficients occur between the (7/2,3,1/2) and (5/2,2,1/2) states, and for the aconitase cluster, the coefficients intermediate between the states (9/2,4,1/2) and (7/2,3,1/2).

An analysis of the magnitude and signs of Fe internal magnetic fields and other Mosssbauer/EPR data for the redox state P(OX) of the P-cluster of nitrogenase helps arrive at a consistent picture of oxidation states and spin coupling patterns in all four observed P-cluster redox states. The 8Fe cluster is made of two (Fe4S4) units, but the two halves exhibit different spin properties due to the presence of an additional serine ligand at one iron site. One cluster has a spin of 1/2 and other 7/2 and both spins are coupled parallel to give a total spin of 4.

The eight protons of the four thiolate CH2 groups and protein ENDOR spectroscopy in single crystals of the synthetic compound (N(C2D5))2 (Fe4S4(SCH2C6D5)4) were used to study the distribution of the unpaired electron spin population in a paramagnetic (Fe4S4)3+ core as a result of gamma irradiation in the crystals. Unlike previous indications of the spin-state being 9/2, 4, 1/2, paramagnetic is near the 7/2, 3, 1/2 state, with the last one signifying the subsequent spin of the cluster, the second, the spin state of the ferric pair and the first, the spin state of the mixed-valence pair. A model determines the law between the isotropic couplings and two indicators, an angular one determining the orientation of every CH bond, and a magnetic one determining the adjacent irons spin population.

The crystal structure of the Escherichia coli O2-sensitive C19G [NiFe]-hydrogenase-1 variant shows that the mutation results in a novel FeS cluster, proximal to the Ni–Fe active site. While the proximal cluster of the native O2-tolerant enzyme can transfer two electrons to that site, EPR spectroscopy shows that the modified cluster can transfer only one electron, this shortfall coinciding with O2 sensitivity. Computational studies on electron transfer help to explain how the structural and redox properties of the novel FeS cluster modulate the observed phenotype. [ABSTRACT FROM AUTHOR]

(57)Fe Q-band ENDOR has been used to study the [4Fe-4S](1+) state created by gamma irradiation of single crystals of the synthetic model compound [N(C(2)H(5))(4)](2)[Fe(4)S(4)(SCH(2)C(6)H(5))(4)] enriched in (57)Fe. This compound is an excellent biomimetic model of the active sites of many 4 iron-4 sulfur proteins, enabling detailed and systematic studies of its oxidized [4Fe-4S](3+) and reduced [4Fe-4S](1+) paramagnetic states. Taking advantage of the fact that Q-band ENDOR, in contrast with X-Band ENDOR, allows for a very good separation of the (57)Fe transitions from those of the protons, the complete hyperfine tensors of the four iron atoms for the [4Fe-4S](1+) species has been measured with precision. For each iron atom, the electron orbital and electron spin isotropic contributions have been determined separately. Moreover, it is remarkable that two (57)Fe hyperfine tensors attributed to the ferrous pair of iron atoms are very different. In effect, one tensor presents a much larger anisotropic part and a much smaller isotropic part than those of the other. This difference has been interpreted in terms of a differential electron orbital hyperfine interaction among the two ferrous ions.