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.
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June 1998
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Research Article|
June 01 1998
Haem insertion, dimerization and reactivation of haem-free rat neuronal nitric oxide synthase
Benjamin HEMMENS;
Benjamin HEMMENS
*Institut für Pharmakologie und Toxikologie, Karl-Franzens Universität Graz, Universitätsplatz 2, A-8010 Graz, Austria
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Antonius C. F. GORREN;
Antonius C. F. GORREN
*Institut für Pharmakologie und Toxikologie, Karl-Franzens Universität Graz, Universitätsplatz 2, A-8010 Graz, Austria
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Kurt SCHMIDT;
Kurt SCHMIDT
*Institut für Pharmakologie und Toxikologie, Karl-Franzens Universität Graz, Universitätsplatz 2, A-8010 Graz, Austria
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Ernst R. WERNER;
Ernst R. WERNER
†Institut für Medizinische Chemie und Biochemie, Universität Innsbruck, Fritz-Pregl-Strasse 3, A-6020 Innsbruck, Austria
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Bernd MAYER
Bernd MAYER
1
*Institut für Pharmakologie und Toxikologie, Karl-Franzens Universität Graz, Universitätsplatz 2, A-8010 Graz, Austria
1To whom correspondence should be addressed (e-mail mayer@kfunigraz.ac.at).
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Publisher: Portland Press Ltd
Received:
November 18 1997
Revision Received:
February 10 1998
Accepted:
March 02 1998
Online ISSN: 1470-8728
Print ISSN: 0264-6021
The Biochemical Society, London © 1998
1998
Biochem J (1998) 332 (2): 337–342.
Article history
Received:
November 18 1997
Revision Received:
February 10 1998
Accepted:
March 02 1998
Citation
Benjamin HEMMENS, Antonius C. F. GORREN, Kurt SCHMIDT, Ernst R. WERNER, Bernd MAYER; Haem insertion, dimerization and reactivation of haem-free rat neuronal nitric oxide synthase. Biochem J 1 June 1998; 332 (2): 337–342. doi: https://doi.org/10.1042/bj3320337
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