Intersubunit intraprotein electron transfer (IET) from flavin mononucleotide (FMN) to heme is essential in nitric oxide (NO) synthesis by NO synthase (NOS). Previous crystal structures and functional studies primarily concerned an enzyme conformation, which serves as the input state for reduction of FMN by electrons from NADPH and flavin adenine dinucleotide (FAD) in the reductase domain. To favor the formation of the output state for the subsequent IET from FMN to heme in the oxygenase domain, a novel truncated two-domain oxyFMN construct of rat neuronal NOS (nNOS), in which only the FMN and heme domains were present, was designed and expressed. The kinetics of IET between the FMN and heme domains in the nNOS oxyFMN construct in the presence and absence of added calmodulin (CaM) were directly determined using laser flash photolysis of CO dissociation in comparative studies on partially reduced oxyFMN and single-domain heme oxygenase constructs. The IET rate constant in the presence of CaM (262 s -1 ) was increased approximately 10-fold compared to that in the absence of CaM (22 s -1 ). The effect of CaM on interdomain interactions was further evidenced by electron paramagnetic resonance (EPR) spectra. This work provides the first direct evidence of the CaM control of electron transfer (ET) between FMN and heme domains through facilitation of the FMN/heme interactions in the output state. Therefore, CaM controls IET between heme and FMN domains by a conformational gated mechanism. This is essential in coupling ET in the reductase domain in NOS with NO synthesis in the oxygenase domain.
Intersubunit intramolecular electron transfer (IET) from FMN to heme is essential in the delivery of electrons required for O2 activation in the heme domain and the subsequent nitric oxide (NO) synthesis by NO synthase (NOS). Previous crystal structures and functional studies primarily concerned an enzyme conformation that serves as the input state for reduction of FMN by electrons from NADPH and FAD in the reductase domain. To favor formation of the output state for the subsequent IET from FMN to heme in the oxygenase domain, a novel truncated two-domain oxyFMN construct murine inducible nitric oxide synthase (iNOS), in which only the FMN and heme domains were present, was designed and expressed. The kinetics of the IET between the FMN and heme domains in this construct was directly determined using laser flash photolysis of CO dissociation in comparative studies on partially reduced oxyFMN and single domain heme oxygenase constructs.
The iron(III) complex of protoporphyrin IX is the basic component of the active sites of cytochromes P-450 and related mono oxygenases. The axial ligand in cytochromes P-450 is a cysteine thiolate, whereas in peroxidases it is the imidazole of a histidine residue of the protein. During the reaction sequence of peroxidase, the iron(Ill) porphyrin moiety of peroxidase reacts with H 2 02 and other mono oxygen donors to give compound I, which, on further one-electron transformation, leads to the formation of an iron(IV) oxo species, compound II of peroxidase. The formation of iron-oxo intermediates, similar to compound I, have been proposed for cytochromes P-450 catalyzed oxidations, except that the axial thiolate ligand favours the formation of the high-valent iron(V) species. Iron-porphyrins and different mono oxygen donors mimic the different reactions of cytochromes P-450 and other mono oxygenases. The formation of the M-O bonds from different metalloporphyrins and mono oxygen donors has been proposed in the hydroxylation of 1,3-dimethylpyrimidines, the epoxidation of aldrin and related olefins as well as in the oxidation of various sulphur compounds. The formation of M-N and M-C bonds has been proposed during the oxidation of different alkylhydrazines and related compounds. The formation of different intermediates and final products are briefly highlighted in the presentation.
Photoinduced inter-and intramolecular electron transfer reactions of carboxylatopentaamminecobalt(lll) and excited state tris(2,2'bipyridine) ruthenium(ll) complexes
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