Structural similarities between the N-terminal domain of Clostridium pasteurianum hydrogenase and plant-type ferredoxins

An N-terminal domain of Clostridium pasteurianum hydrogenase I, encompassin g 76 residues out of the 574 composing the full-size enzyme, had previously been overproduced in Escherichia coli and shown to form a stable fold arou nd a [2Fe-2S] cluster. This domain displays only marginal sequence similari ty with [2Fe-2S] proteins of known structure, and therefore, two-dimensiona l H-1 NMR has been implemented to elucidate features of the polypeptide fol d. Despite the perturbing presence of the paramagnetic [2Fe-2S] cluster, 57 spin systems were detected in the TOCSY spectra, 52 of which were sequenti ally assigned through NOE connectivities. Several secondary structure eleme nts were identified. The N terminus of the protein consists of two antipara llel beta strands followed by an alpha helix contacting both strands. Two a dditional antiparallel beta strands, one of them at the C terminus of the s equence, form a four-stranded beta sheet together with the two N-terminal s trands. The proton resonances that can be attributed to this beta 2 alpha b eta 2 structural motif undergo no paramagnetic perturbations, suggesting th at it is distant from the [2Fe-2S] cluster. In plant- and mammalian-type fe rredoxins, a very similar structural pattern is found in the part of the pr otein farthest from the [2Fe-2S] cluster. This indicates that the N-termina l domain of C. pasteurianum hydrogenase folds in a manner very similar to t hose of plant- and mammalian-type ferredoxins over a significant part (ca. 50%) of its structure. Even in the vicinity of the metal site, where H-1 NM R data are blurred by paramagnetic interactions, the N-terminal domains of hydrogenase and mammalian- and plant-type ferredoxins most likely display s ignificant structural similarity, as inferred from local sequence alignment s and from previously reported circular dichroism and resonance Raman spect ra. These data afford structural information on a kind of [2Fe-2S] cluster- containing domain that occurs in a number of redox enzymes and complexes. I n addition, together with previously published sequence alignments, they hi ghlight the widespread distribution of the plant-type ferredoxin fold in bi oenergetic systems encompassing anaerobic metabolism, photosynthesis, and a erobic respiratory chains.