Evidence for oxidation-state-dependent conformational changes in human ferredoxin from multinuclear, multidimensional NMR spectroscopy.

Human ferredoxin belongs to the vertebrate ferredoxin family which includes bovine adrenodoxin. It is a small (13.8 kDa) acidic protein with a [2Fe-2S] cluster. It functions as an electron shuttle in the cholesterol side-chain cleavage reaction which is the first step of steroid hormone biosynthesis. The protein studied here was produced in Escherichia coli and doubly labeled with 13C and 15N. The diamagnetic 15N, 13C', 13C alpha, 13C beta, 1H alpha, and 1HN resonances from about 70% of the 124 amino acid residues for oxidized human ferredoxin and 80% of those for the reduced protein have been assigned primarily on the basis of results from three-dimensional, triple-resonance experiments. Secondary structure features for human ferredoxin in its oxidized and reduced states have been identified from a combination of chemical shift index and NOE data. Comparison of NMR results from the protein in its oxidized and reduced states indicates that structural changes accompany the change in the oxidation state of the [2Fe-2S] cluster. Major differences are localized at two regions: residues 29-31 and residues 109-124; the latter stretch forms the C-terminal region of the protein. The possible functional significance of these oxidation-state-dependent structural changes is discussed.