TY - JOUR
T1 - Assembly and activation of heme-deficient neuronal NO synthase with various porphyrins
AU - Bender, Andrew T.
AU - Kamada, Yasuhiko
AU - Kleaveland, Patricia A.
AU - Osawa, Yoichi
N1 - Funding Information:
This investigation was supported by National Institutes of Health Grants ES08365 (to Y.O.) and a Predoctoral Fellowship from the Pharmaceutical Research and Manufacturers of America Foundation (to A.T.B.). Y.O. is the recipient of an Established Investigator Award from the American Heart Association. We thank Solomon Snyder for providing cDNA used in this work.
PY - 2002/9/20
Y1 - 2002/9/20
N2 - 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.
AB - 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.
KW - Assembly
KW - Dimerization
KW - Heme
KW - Hemoprotein
KW - Nitric oxide
KW - Nitric oxide synthase
KW - Porphyrin
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U2 - 10.1016/S0162-0134(02)00430-0
DO - 10.1016/S0162-0134(02)00430-0
M3 - Article
C2 - 12237228
AN - SCOPUS:0037145074
VL - 91
SP - 625
EP - 634
JO - Journal of Inorganic Biochemistry
JF - Journal of Inorganic Biochemistry
SN - 0162-0134
IS - 4
ER -