Assembly and activation of heme-deficient neuronal NO synthase with various porphyrins.
The heme prosthetic group of NO synthase is critical for catalytic activity as well as assembly of the enzyme to the native homodimeric form. In the current study, we examined if structurally different metal porphyrins could substitute for the native heme prosthetic group in neuronal NO synthase (nNOS) with regard to assembly and catalysis. We established, with the use of a recently developed in vitro method that functionally reconstitutes heme-deficient apo-nNOS, that Fe-mesoporphyrin IX or Fe-deuteroporphyrin IX can substitute for heme and lead to assembly of a functional nNOS, albeit with lower activity. Fe-protoporphyrin IX dimethyl ester or the metal free protoporphyrin IX, however, lead to minimal assembly of nNOS. Protoporphyrin IX compounds where the native Fe was substituted with Zn, Mn, Co, or Sn lead to assembly of nNOS, but no detectable NO was synthesized in the presence of NADPH and L-arginine. Thus, the presence of the metal and propionic acid groups, but not the vinyl moieties, of heme are important structural features in assembly of nNOS. These studies establish that the mechanism of assembly and catalysis of nNOS can be probed with structurally diverse metal porphyrins.
Bender
Andrew T
AT
Department of Pharmacology, The University of Michigan Medical School, Ann Arbor, MI 48109-0632, USA.
Kamada
Yasuhiko
Y
Kleaveland
Patricia A
PA
Osawa
Yoichi
Y
D000818:Animals; D001051:Apoenzymes / Q000737:chemistry / Q000378:metabolism; D001665:Binding Sites; D002412:Cations; D002460:Cell Line; D019281:Dimerization; D004789:Enzyme Activation; D006418:Heme / Q000172:deficiency; D006801:Humans; D019001:Nitric Oxide Synthase / Q000737:chemistry / Q000378:metabolism*; D052248:Nitric Oxide Synthase Type I; D011166:Porphyrins / Q000737:chemistry / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013053:Spectrophotometry; D018411:Spodoptera; D014162:Transfection
D001051:Apoenzymes; D002412:Cations; D011166:Porphyrins; D011994:Recombinant Proteins; D006418:Heme; C496320:NOS1 protein, human; D019001:Nitric Oxide Synthase; D052248:Nitric Oxide Synthase Type I