HEME INSERTION, DIMERIZATION AND REACTIVATION OF HEME-FREE RAT NEURONAL NITRIC-OXIDE SYNTHASE

The nitric oxide synthases are dimeric enzymes in which the intersubun it contacts are formed by the P-450-haem-containing, tetrahydrobiopter in-dependent oxygenase domain. The dimerization of the neuronal isoenz yme was shown previously to be triggered by Fe-protoporphyrin IX (haem in). We report for the first time the reactivation of the haem-deficie nt neuronal isoenzyme (from rat, expressed in a baculovirus/insect cel l system) after haem reconstitution. We further examined the reconstit ution of the enzyme with protoporphyrin IX (PPIX) and its Mn and Co co mplexes. All of these porphyrins inserted into the haem pocket, as ass essed by quenching of intrinsic protein fluorescence. In addition to h aemin, MnPPIX stimulated dimerization, as measured by gel filtration a nd by cross-linking with glutaraldehyde. In contrast, neither CoPPIX n or PPIX stimulated dimerization. The absorbance spectra of the reconst ituted enzymes were measured and compared with published results on P- 450 enzymes reconstituted with the same metals. The results suggest th at those metalloporphyrins which caused dimerization were able to acqu ire a thiolate ligand from the protein, and we propose that this ligat ion is the trigger for dimerization. Substrate and tetrahydrobiopterin binding sites only emerged with the metalloporphyrins that caused dim erization.