A new electron transport mechanism in mitochondrial steroid hydroxylase systems based on structural changes upon the reduction of adrenodoxin.

The adrenal ferredoxin (adrenodoxin, Adx) is an acidic 14.4-kDa [2Fe-2S] ferredoxin that belongs to the vertebrate ferredoxin family. It is involved in the electron transfer from the flavoenzyme NADPH-adrenodoxin-reductase to cytochromes P-450(scc) and P-450(11)(beta). The interaction between the redox partners during electron transport has not yet been fully established. Determining the tertiary structure of an electron-transfer protein may be very helpful in understanding the transport mechanism. In the present work, we report a structural study on the oxidized and reduced forms of bovine adrenodoxin (bAdx) in solution using high-resolution NMR spectroscopy. The protein was produced in Escherichia coli and singly or doubly labeled with (15)N or (13)C/(15)N, respectively. Approximately 70 and 75% of the (15)N, (13)C, and (1)H resonances could be assigned for the reduced and the oxidized bAdx, respectively. The secondary and tertiary structures of the reduced and oxidized states were determined using NOE distance information. (1)H(N)-T(1) relaxation times of certain residues were used to obtain additional distance constraints to the [2Fe-2S] cluster. The results suggest that the solution structure of oxidized Adx is quite similar to the X-ray structure. However, structural changes occur upon reduction of the [2Fe-2S] cluster, as indicated by NMR measurements. It could be shown that these conformational changes, especially in the C-terminal region, cause the dissociation of the Adx dimer upon reduction. A new electron transport mechanism proceeding via a modified shuttle mechanism, with both monomers and dimers acting as electron carriers, is proposed.