Saccharomyces, human and two Arabidopsis (ATR1 and ATR2) NADPH-P-450 reductases were expressed in yeast, purified to homogeneity and used to raise antibodies. Among the P-450-reductases, ATR2 contrasted by its very low FMN affinity and required a thiol-reducing agent for efficient cofactor binding to the FMN-depleted enzyme. Analysis of reductase kinetic properties using artificial acceptors and different salt conditions suggested marked differences between reductases in their FAD and FMN environments and confirmed the unusual properties of the ATR2 FMN-binding domain. Courses of flavin reductions by NADPH were analysed by rapid kinetic studies. The human enzyme was characterized by a FAD reduction rate sixfold to tenfold slower than values for the three other reductases. Following the fast phase of reduction, expected accumulation of flavin semiquinone was observed for the human and ATR1 but not for ATR2 and the yeast reductases. Consistently, redox potential for the FMN semiquinone/reduced couple in the yeast enzyme was found to be more positive than the value for the FMN oxidized/semiquinone couple. This situation was reminiscent of similar inversion observed in bacterial P-450 BM3 reductase. Affinities of reductases for rabbit P-450 2B4 and supported monooxygenase activities in reconstituted systems highly depended on the reductase source. The human enzyme exhibited the highest affinity but supported the lowest kcat whereas the yeast reductase gave the best kcat but with the lowest affinity. ATR1 exhibited both high affinity and efficiency. No simple relation was found between reductase activities with artificial and natural (P-450) acceptors. Thus marked differences in kinetic and redox parameters between reductases dramatically affect their respective abilities to to support P-450 functions.
| Predicate | Object |
|---|---|
| rdf:type | |
| rdfs:comment |
Saccharomyces, human and two Arabidopsis (ATR1 and ATR2) NADPH-P-450 reductases were expressed in yeast, purified to homogeneity and used to raise antibodies. Among the P-450-reductases, ATR2 contrasted by its very low FMN affinity and required a thiol-reducing agent for efficient cofactor binding to the FMN-depleted enzyme. Analysis of reductase kinetic properties using artificial acceptors and different salt conditions suggested marked differences between reductases in their FAD and FMN environments and confirmed the unusual properties of the ATR2 FMN-binding domain. Courses of flavin reductions by NADPH were analysed by rapid kinetic studies. The human enzyme was characterized by a FAD reduction rate sixfold to tenfold slower than values for the three other reductases. Following the fast phase of reduction, expected accumulation of flavin semiquinone was observed for the human and ATR1 but not for ATR2 and the yeast reductases. Consistently, redox potential for the FMN semiquinone/reduced couple in the yeast enzyme was found to be more positive than the value for the FMN oxidized/semiquinone couple. This situation was reminiscent of similar inversion observed in bacterial P-450 BM3 reductase. Affinities of reductases for rabbit P-450 2B4 and supported monooxygenase activities in reconstituted systems highly depended on the reductase source. The human enzyme exhibited the highest affinity but supported the lowest kcat whereas the yeast reductase gave the best kcat but with the lowest affinity. ATR1 exhibited both high affinity and efficiency. No simple relation was found between reductase activities with artificial and natural (P-450) acceptors. Thus marked differences in kinetic and redox parameters between reductases dramatically affect their respective abilities to to support P-450 functions.
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| skos:exactMatch | |
| uniprot:name |
Eur. J. Biochem.
|
| uniprot:author |
Louerat-Oriou B.,
Perret A.,
Pompon D.
|
| uniprot:date |
1998
|
| uniprot:pages |
1040-1049
|
| uniprot:title |
Differential redox and electron-transfer properties of purified yeast, plant and human NADPH-cytochrome P-450 reductases highly modulate cytochrome P-450 activities.
|
| uniprot:volume |
258
|
| dc-term:identifier |
doi:10.1046/j.1432-1327.1998.2581040.x
|