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The three-dimensional structure of a truncated mutant of bovine adrenodoxin has been resolved at 1.85 A resolution by MAD. The protein consists of a large core region and a more flexible hairpin loop bearing residues which have been previously described as being involved in redox partner recognition. To study the role of distinct protein domains and amino acids of adrenodoxin in interaction with adrenodoxin reductase (AdR), CYP11A1 and CYP11B1, as well as in electron transfer, mutants of adrenodoxin have been prepared by site-directed mutagenesis and produced in Escherichia coli, and their structural and functional properties have been characterized in detail. It could be demonstrated that Tyr82 is located at the edge of the flexible interaction loop of adrenodoxin participating in interactions with AdR and P450s. His56, being close to Tyr82, forms a bridge between the core region of adrenodoxin and the interaction loop. Its role in transmitting changes of the cluster region to the interaction site has also been supported by functional studies. Pro108 of adrenodoxin, the only proline residue contained in the protein and being conserved in this position among several other vertebrate-type ferredoxins, has been demonstrated to be of importance for the correct folding of this protein.

We expressed a guinea pig 11 beta-hydroxylase cDNA (1) in COS-1 cells. In order to find the optimal expression system we compared three expression plasmids, each driven by a different promoter. Although promoters exhibited different transcriptional activities this did not result in different enzymatic activities. Upon cotransfection with bovine adrenodoxin a 5-fold increase of enzyme activity was achieved. A comparison with the bovine 11 beta-hydroxylase clearly demonstrated that the guinea pig enzyme was not able to produce significant amounts of 18-hydroxylated and 18-oxidized products from deoxycorticosterone under the experimental conditions used.

The cDNAs of human 11 beta hydroxylase (CYP11B1) and aldosterone synthase (CYP11B2) were cloned from surgically removed normal human adrenal using polymerase chain reaction. The cloned cDNAs were transfected into COS-7 cells and steroid hydroxylase activity of the two cytochromes P450 expressed was determined in order to elucidate their the functional characteristics. Stable expression of human CYP11B1 or CYP11B2 was performed in V79 hamster cells and confirmed by Southern blotting, Northern blotting, and enzymatic activity. Interestingly, recombinant V79 cells were able to support CYP11B1 and CYP11B2 dependent steroid conversion without additional heterologous expression of the corresponding electron donor system.