Putidaredoxin reductase, a new function for an old protein.
Properties of recombinant wild type (WT) and six-histidine tag-fused (His(6)) putidaredoxin reductase (Pdr), a FAD-containing component of the soluble cytochrome P450cam monooxygenase system from Pseudomonas putida, have been studied. Both WT and His(6) Pdr were found to undergo a monomer-dimer association-dissociation and were partially present as an NAD(+)-bound form. Although molecular, spectral, and electron transferring properties of recombinant His(6) Pdr to artificial and native electron acceptors were similar to those of the WT protein, the presence of eight additional C-terminal amino acid residues, Pro-Arg-His-His-His-His-His-His, had a crucial effect on the enzyme interaction with oxidized pyridine nucleotide. Under anaerobic conditions, NAD(+) induced in His(6) Pdr spectral changes indicative of flavin reduction and formation of the charge transfer complex between the reduced FAD and NAD(+). The reaction proceeded considerably faster in the presence of free histidine and thiol-reducing agents, such as dithiothreitol and reduced glutathione. In the presence of any of these three reagents, NAD(+) was capable of inducing reduction of the flavin in WT Pdr. Free thiol groups were identified as an internal source of electrons in the enzyme. The results showed that WT and His(6) Pdr were able to function as NAD(H)-dependent dithiol/disulfide oxidoreductases catalyzing both forward and reverse reactions, NAD(+)-dependent oxidation of thiols, and NADH-dependent reduction of disulfides. This function of the flavoprotein can be dissociated from electron transfer to putidaredoxin. Similarity of Pdr to the enzymes of the glutathione reductase family is discussed.
Sevrioukova
Irina F
IF
Department of Molecular Biology and the Program in Macromolecular Structure, University of California, Irvine 92612-3900, USA. sevrioui@uci.edu
Poulos
Thomas L
TL
D001483:Base Sequence; D017931:DNA Primers; D005415:Flavins / Q000378:metabolism; D006639:Histidine / Q000378:metabolism; D009247:NADH, NADPH Oxidoreductases / Q000378:metabolism*; D010084:Oxidation-Reduction; D011994:Recombinant Proteins / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013438:Sulfhydryl Compounds / Q000378:metabolism
D017931:DNA Primers; D005415:Flavins; D011994:Recombinant Proteins; D013438:Sulfhydryl Compounds; D006639:Histidine; D009247:NADH, NADPH Oxidoreductases; C022486:putidaredoxin reductase