Iron-chromophore circular dichroism of [Fe]-hydrogenase: the conformational change required for H2 activation.

Circular dichroism (CD) spectroscopy is a very sensitive method used to detect changes in inherently chiral chromophores and in achiral chromophores embedded in chiral surroundings. Information on the secondary structures of proteins can be obtained by CD spectroscopy in the far-UV spectral region (190–250 nm), and CD in the near-UV region (250–350 nm) can be sensitive to certain aspects of tertiary structure. Near-UV and visible CD spectroscopy is also used to analyze metal complexes and the active site of metal-containing enzymes, such as P450 (300–500 nm), galactose oxidase (300–700 nm), and biotin sulfoxide reductase (300–650 nm). The [Fe]-hydrogenase found in many methanogenic archaea catalyzes the reversible reduction of methenyltetrahydromethanopterin (methenyl-H4MPT ) with H2 to methylenetetrahydromethanopterin (methylene-H4MPT) in the methanogenic pathway from CO2 and H2 (Scheme 1). [3] This hydrogenase contains a unique iron guanylyl pyridinol (FeGP) cofactor and lacks iron–sulfur clusters. A model of the iron-complex structure of the FeGP cofactor bound to the enzyme has been proposed on the basis of crystallographic, spectroscopic, and chemical analyses. In the model, the iron ion is ligated with an sp-hybridized nitrogen atom, an acyl carbon atom, the Cys176 sulfur atom, two CO molecules, and one “solvent” molecule to form a chiral iron complex (Scheme 2). Several model compounds of the FeGP cofactor have been synthesized. The crystal structure of the [Fe]-hydrogenase from Methanocaldococcus jannaschii reveals that the homodimer is composed of three domains: two peripheral N-terminal domains to which the FeGP cofactor binds, and one central domain composed of two intertwined C-terminal domains. The peripheral domains and the central domain form the two active-site clefts. In the cocrystal structure of the [Fe]hydrogenase C176A mutant and the substrate methyleneH4MPT, the substrate is bound to the central domain in the open form of the enzyme (see Figure S1 A in the Supporting Information). The distance between the iron center of the FeGP cofactor and the substrate is too far for the iron center and the substrate to interact. Hiromoto et al. predicted that the active-site cleft is closed upon binding of the substrates, and that the iron site moves close to the carbocation of the substrate, which is the hydride acceptor in the reaction. It was also proposed that H2 is supplied to the active site through a narrow channel formed between the peripheral and central domains after closing of the cleft, and that H2 then interacts with methenyl-H4MPT +