Haem insertion, dimerization and reactivation of haem-free rat neuronal nitric oxide synthase.
The nitric oxide synthases are dimeric enzymes in which the intersubunit contacts are formed by the P-450-haem-containing, tetrahydrobiopterin-dependent oxygenase domain. The dimerization of the neuronal isoenzyme was shown previously to be triggered by Fe-protoporphyrin IX (haemin). We report for the first time the reactivation of the haem-deficient neuronal isoenzyme (from rat, expressed in a baculovirus/insect cell system) after haem reconstitution. We further examined the reconstitution of the enzyme with protoporphyrin IX (PPIX) and its Mn and Co complexes. All of these porphyrins inserted into the haem pocket, as assessed by quenching of intrinsic protein fluorescence. In addition to haemin, MnPPIX stimulated dimerization, as measured by gel filtration and by cross-linking with glutaraldehyde. In contrast, neither CoPPIX nor PPIX stimulated dimerization. The absorbance spectra of the reconstituted enzymes were measured and compared with published results on P-450 enzymes reconstituted with the same metals. The results suggest that those metalloporphyrins which caused dimerization were able to acquire a thiolate ligand from the protein, and we propose that this ligation is the trigger for dimerization. Substrate and tetrahydrobiopterin binding sites only emerged with the metalloporphyrins that caused dimerization.
Hemmens
B
B
Institut für Pharmakologie und Toxikologie, Karl-Franzens Universität Graz, Universitätsplatz 2, A-8010 Graz, Austria.
Gorren
A C
AC
Schmidt
K
K
Werner
E R
ER
Mayer
B
B
D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D001665:Binding Sites; D001708:Biopterins / Q000031:analogs & derivatives / Q000378:metabolism; D003432:Cross-Linking Reagents / Q000378:metabolism; D019281:Dimerization; D004789:Enzyme Activation / Q000502:physiology; D005420:Flavoproteins / Q000737:chemistry; D005976:Glutaral / Q000378:metabolism; D006418:Heme / Q000494:pharmacology*; D008345:Manganese / Q000494:pharmacology; D019001:Nitric Oxide Synthase / Q000737:chemistry*; D019335:Nitroarginine / Q000378:metabolism; D011166:Porphyrins / Q000494:pharmacology; D051381:Rats; D011994:Recombinant Proteins / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D013053:Spectrophotometry
D003432:Cross-Linking Reagents; D005420:Flavoproteins; D011166:Porphyrins; D011994:Recombinant Proteins; D019335:Nitroarginine; D001708:Biopterins; D006418:Heme; D008345:Manganese; D019001:Nitric Oxide Synthase; C003402:sapropterin; D005976:Glutaral