Laser flash induced electron transfer in P450cam monooxygenase: putidaredoxin reductase-putidaredoxin interaction.
The P450cam monooxygenase from Pseudomonas putida consists of three redox proteins: NADH-putidaredoxin reductase (Pdr), putidaredoxin (Pdx), and cytochrome P450cam. The redox properties of the FAD-containing Pdr and the mechanism of Pdr-Pdx complex formation are the least studied aspects of this system. We have utilized laser flash photolysis techniques to produce the one-electron-reduced species of Pdr, to characterize its spectral and electron-transferring properties, and to investigate the mechanism of its interaction with Pdx. Upon flash-induced reduction by 5-deazariboflavin semiquinone, the flavoprotein forms a blue neutral FAD semiquinone (FADH(*)). The FAD semiquinone was unstable and partially disproportionated into fully oxidized and fully reduced flavin. The rate of FADH(*) decay was dependent on ionic strength and NAD(+). In the mixture of Pdr and Pdx, where the flavoprotein was present in excess, electron transfer (ET) from FADH(*) to the iron-sulfur cluster was observed. The Pdr-to-Pdx ET rates were maximal at an ionic strength of 0.35 where a kinetic dissociation constant (K(d)) for the transient Pdr-Pdx complex and a limiting k(obs) value were equal to 5 microM and 226 s(-1), respectively. This indicates that FADH(*) is a kinetically significant intermediate in the turnover of P450cam monooxygenase. Transient kinetics as a function of ionic strength suggest that, in contrast to the Pdx-P450cam redox couple where complex formation is predominantly electrostatic, the Pdx-Pdr association is driven by nonelectrostatic interactions.
Sevrioukova
I F
IF
Department of Molecular Biology and Biochemistry, University of California, Irvine, California 92612-3900, USA. sevrioui@uci.edu
Hazzard
J T
JT
Tollin
G
G
Poulos
T L
TL
D019475:Camphor 5-Monooxygenase / Q000378:metabolism*; D004579:Electron Transport; D005288:Ferredoxins / Q000378:metabolism*; D007834:Lasers; D009247:NADH, NADPH Oxidoreductases / Q000378:metabolism*; D010782:Photolysis; D016958:Pseudomonas putida / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism
D005288:Ferredoxins; D011994:Recombinant Proteins; C100137:putidaredoxin; D019475:Camphor 5-Monooxygenase; D009247:NADH, NADPH Oxidoreductases; C022486:putidaredoxin reductase