On the structure of putidaredoxin and cytochrome P-450 cam and their mode of interaction.

Structural and immunochemical experiments with putidaredoxin, cytochrome P-450cam, and their 1:1 complex have led us to the following conclusions: Despite the remarkable sequence homology between putidaredoxin and adrenodoxin which permits a tentative assignment of cysteines binding to the (Fe-S)2 prosthetic group, these redox proteins cannot replace each other in reconstitution experiments because putidaredoxin contains a disulfide loop close to its P-450cam binding site. This feature may also be responsible for the complete lack of immunochemical cross reactivity between these proteins. The stability of putidaredoxin can be enhanced significantly by cross linkage with glutaraldehyde without change in spectral, catalytic, or immunochemical properties, Putidaredoxin also gains stability by binding to the P-450-camphor complex in a 1:1 ratio. Precipitation of this complex with anti-P-450cam antibodies gives access to site specific antibodies directed against the putidaredoxin binding site of P-450cam. A series of putidaredoxin-cytochrome P-450cam-substrate complexes with ratios of 1 to 6 molecules of redoxin per molecule of cytochrome have been obtained by migration of excess redoxin across prefocused P-450cam in electrofocusing. Complete inhibition of camphor hydroxylation was achieved by anti-P-450cam antibodies, their Fab fragments, anti-putidaredoxin-trimer antibodies, and antibodies directed against the putidaredoxin-P-450cam complex. Five major antigenic sites were tentatively established for P-450cam, two of which seem to be associated with the BrCN hemepeptide while one each relates to the putidaredoxin binding site, the Trp-Arg site close to the C-terminus, and the site surrounding the most reactive SH group which gives rise to dimer formation. Iodination, of P-450cam at tyrosyl residues only permitted use of a sensitive radioimmunoassay procedure for testing of cross reacting material (CRM) remaining after degradation of P-450cam with BrCN and enzymes, denaturation with acetone, and complex formation with the redoxin. The BrCN hemepeptide still has a Soret maximum at 390 nm and reacts with CO yielding a P-420 spectrum. All 6 half-cystines of P-450cam are present as free sulfhydryls and can be titrated after denaturation but only 4 of them are available in the P-450-camphor complex. Three of these are close to each other and the heme, and work in concert; their alkylation with N-ethyl maleimide (NEM) leads to shifts of the Soret from 391 to 417 nm and concomitant changes in redox potential, EPR-signals and DPNH-reactivity. The fifth SH group is protected by camphor while the 6th SH group, still present in the BrCN heme-peptide, is implicated in chelation to the heme iron by a drastic change in EPR spectra, reflecting pure axial symmetry at the heme after complete alkylation by NEM.