cubane: i.s. ¼ 0.47, 0.47, q.s. ¼ 0.85, 1.35; di-iron subsite:i.s. ¼ 0.08, q.s. ¼ 0.87 mm s21) measured at 4.2 K. This is concor-dant with the generally accepted re-assignment of the electronicstructure of the six-iron core of the reduced biological cluster as[{Fe(I).Fe(I)}subsite-{4Fe4S}cubane2þ]29, the established redoxconfiguration of our synthetic cluster. In the synthetic system, wehave seen that the reduction of the cubane unit by one electron to the{4Fe4S}þlevel is easier than is reduction of the {Fe(I).Fe(I)}subsite,which is coordinatively saturated with a closed-shell (36-electron)configuration. A corresponding state of the H-cluster, in which thecubane unit is reduced to the {4Fe4S}þlevel, has yet to be detected,although neighbouring {4Fe4S} relay centres in C. pasteurianumhydrogenase II can be reduced to this level29. This raises the questionas to whether the {4Fe4S}þlevel of the H-cluster is physiologicallyaccessed during turnover. A vacant or (weakly) water-coordinatedsite has been identified crystallographically in the enzyme at thedistal iron atom in the resting state of the enzym e. It is possible thatprotonation at this site lowers the energy of the Fe(I)·Fe(I) subsiteunit sufficiently to enable its reduction by the anchored cubaneoperating at the {4Fe4S}2þlevel. That D is capable of electrocatalys-ing proton reduction may be similarly linked to the formation of avacant site, in this case by th e opening of them-S bridge onreduction.The artificial H-clusters reported here should enhance our under-standing of the intimate chemistry of the natural process, and lead tosystems with low overpotentials for hydrogen uptake/evolution11,28.Given that redox-active {4Fe4S}2þ-centres can be incorporated athigh concentration into cysteine functionalized electropolymers30,we can envisage their modification, using the chemistry we havedescribed, thereby providing a route to advanced electrodematerials. AReceived 14 October; accepted 14 December 2004; doi:10.1038/nature03298.1. Peters, J. W., Lanzilotta, W. N., Lemon, B. J. & Seefeldt, L. C. X-ray crystal structure of the Fe-onlyhydrogenase (Cpl) from Clostridium pasteurianum to 1.8 angstrom resolution. Science 282,1853–1858 (1998).2. Nicolet, Y., Piras, C., Legrand, P., Hatchikian, C. E. & Fontecilla-Camps, J. C. Desulfovibriodesulfuricans iron hydrogenase: the structure shows unusual coordination to an active site Febinuclear center. Struct. Fold. Des. 7, 13–23 (1999).3. Nicolet, Y., Lemon, B. J., Fontecilla-Camps, J. C. & Peters, J. W. A novel FeS cluster in Fe-onlyhydrogenases. Trends Biochem. Sci. 25, 138–143 (2000).4. Armstrong, F. A. Hydrogenases: active site puzzles and progress. Curr. Opin. Chem. Biol. 8, 133–140(2004).5. Evans, D. J. & Pickett, C. J. Chemistry and the hydrogenases. Chem. Soc. Rev. 32, 268–275 (2003).6. Lyon, E. J., Georgakaki, I. P., Reibenspies, J. H. & Darensbourg, M. Y. Carbon monoxide and cyanideligands in a classical organometallic complex model for Fe-only hydrogenase. Angew. Chem. Int. Edn38, 3178–3180 (1999).7. Schmidt, M., Contakes, S. M. & Rauchfuss, T. B. First generation analogues of the binuclear site in theFe-only hydrogenases: Fe2(m-SR)2(CO)4(CN)222. J. Am. Chem. Soc. 121, 9736–9737 (1999).8. Le Cloirec, A. et al. A di-iron dithiolate possessing structural elements of the carbonyl/cyanide sub-siteof the H-centre of Fe-only hydrogenase. Chem. Commun. 2285–2286 (1999).9. Razavet, M. et al. Transient FTIR spectroelectrochemical and stopped-flow detection of a mixedvalence {Fe(I)-Fe(II)} bridging carbonyl intermediate with structural elements and spectroscopiccharacteristics of the di-iron sub-site of all-iron hydrogenase. Chem. Commun. 700–701 (2002).10. George, S. J., Cui, Z., Razavet, M. & Pickett, C. J. The di-iron subsite of all-iron hydrogenase:Mechanism of cyanation of a synthetic {2Fe3S} – carbonyl assembly. Chem. Eur. J. 8, 4037–4046(2002).11. Gloaguen, F., Lawrence, J. D., Rauchfuss, T. B., Benard, M. & Rohmer, M. M. Bimetallic carbonylthiolates as functional models for Fe-only hydrogenases. Inorg. Chem. 41, 6573–6582 (2002).12. Ott, S., Kritikos, M., Akermark, B., Sun, L. C. & Lomoth, R. A biomimetic pathway for hydrogenevolution from a model of the iron hydrogenase active site. Angew. Chem. Int. Edn 43, 1006–1009(2004).13. Cao, Z. X. & Hall, M. B. Modeling the active sites in metalloenzymes. 3. Density functionalcalculations on models for Fe-hydrogenase: Structures and vibrational frequencies of the observedredox forms and the reaction mechanism at the diiron active center. J. Am. Chem. Soc. 123, 3734–3742(2001).14. Liu, Z. P. & Hu, P. A densit y functional theory study on the active center of Fe-only hydrogenase:Characterization and electronic structure of the redox states. J. Am. Chem. Soc. 124, 5175–5182(2002).15. Bruschi, M., Fantucci, P. & De Gioia, L. Density functional theory investigation of the active site ofFe-hydrogenases. Systematic study of the effects of redox state and ligands hardness on structural andelectronic properties of complexes related to the [2Fe]Hsubcluster. Inorg. Chem. 43, 3733–3741(2004).16. Basic Research Needs for the Hydrogen Economy (Report of the Basic Energy Sciences Workshop onHydrogen Production, Storage, and Use, 13–15 May 2003, Office of Science, US Department ofEnergy); available at khttp://www.eere.energy.gov/hydrogenandfuelcells/pdfs/bes_project.pdfl.17. Platinum and Hydrogen for Fuel Cell Vehicles (UK Department for Transport, September 2003);available at khttp://www.dft.gov.uk/stellent/groups/dft_roads/documents/page/dft_roads_024056.hcspl.18. Reihlen, H., Gruhl, A. & Hessling, G. U¨ber den photochemischen und oxydativen Abbau vonCarbonylen. Liebigs Ann. Chem. 472, 268–287 (1929).19. Razavet, M. et al. All-iron hydrogenase: synthesis, structure and properties of {2Fe3S}-assembliesrelated to the di-iron sub-site of the H-cluster. Dalton Trans. 586–595 (2003).20. Stack, T. D. P. & Holm, R. H. Subsite-specific functionalization of the [4Fe-4S]2þanalog of iron sulfurprotein clusters. J. Am. Chem. Soc. 109, 2546–2547 (1987).21. Perdew, J. P. Density-functional approximation for the correlation-energy of the inhomogeneouselectron-gas. Phys. Rev. B 33, 8822–8824 (1986).22. Becke, A. D. Density-functional exchange-energy approximation with correct asymptotic-behavior.Phys. Rev. A. 38, 3098–3100 (1988).23. Schafer, A., Huber, C. & Ahlrichs, R. Fully optimized contracted Gaussian-basis