Heterologous biosynthesis and characterization of the [2Fe-2S]-containing N-terminal domain of Clostridium pasteurianum hydrogenase.
The primary structure of Clostridium pasteurianum hydrogenase I appears to be composed of modules suggesting that the various iron-sulfur clusters present in this enzyme might be segregated in structurally distinct domains. On the basis of this observation, a gene fragment encoding the 76 N-terminal residues of this enzyme has been expressed in Escherichia coli. The polypeptide thus produced contains a [2Fe-2S]n+ cluster of which the oxidized level (n = 2) has been monitored by UV-visible absorption, circular dichroism, and resonance Raman spectroscopy. This cluster can be reduced by dithionite or electrochemically to the n = 1 level which has been investigated by EPR and by low-temperature magnetic circular dichroism. The redox potential of the +2 to +1 transition is -400 mV (vs the normal hydrogen electrode). The spectroscopic and redox results indicate a [2Fe-2S]2+/+ chromophore coordinated by four cysteine ligands in a protein fold similar to that found in plant- and mammalian-type ferredoxins. Among the five cysteines present in the N-terminal hydrogenase fragment, four (in positions 34, 46, 49, and 62) are conserved in other sequences and are therefore the most likely ligands of the [2Fe-2S] site. The fifth cysteine, in position 39, can be dismissed on the grounds that the Cys39Ala mutation does not alter any of the properties of the iron-sulfur cluster. The spectroscopic signatures of this chromophore are practically identical with some of those reported for full-size hydrogenase. This confirms that C. pasteurianum hydrogenase I contains a [2Fe-2S] cluster and indicates that the polypeptide fold around the metal site of the N-terminal fragment is very similar, if not identical, to that occurring in the full-size protein. The N-terminal sequence of this hydrogenase is homologous to sequences of a number of proteins or protein domains, including a subunit of NADH-ubiquinone oxidoreductase of respiratory chains. From that, it can be anticipated that the structural domain isolated and described here is a building block of electron transfer complexes involved in various bioenergetic processes.
Atta
M
M
Département de Biologie Moléculaire et Structurale, CEA-Grenoble, France.
Lafferty
M E
ME
Johnson
M K
MK
Gaillard
J
J
Meyer
J
J
D000595:Amino Acid Sequence; D003013:Clostridium / Q000201:enzymology*; D004578:Electron Spin Resonance Spectroscopy; D004926:Escherichia coli / Q000235:genetics; D005288:Ferredoxins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D006864:Hydrogenase / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D010446:Peptide Fragments / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification; D013056:Spectrophotometry, Ultraviolet; D013059:Spectrum Analysis, Raman
D005288:Ferredoxins; D010446:Peptide Fragments; D011994:Recombinant Proteins; D006864:Hydrogenase