Putidaredoxin Reductase, a New Function for an Old Protein

Abstract

Properties of recombinant wild type (WT) and sixhistidine 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 associationdissociation 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-HisHis-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.A FAD-containing putidaredoxin reductase (Pdr) 1 catalyzes the transfer of electrons from NADH to an iron-sulfur protein, putidaredoxin (Pdx), in the cytochrome P450cam monooxygenase system from Pseudomonas putida (1). Two-electron reduction of FAD with NADH is followed by the transfer of two single electrons from the flavin to Pdx. From Pdx, electrons are finally transferred to the terminal oxygenase cytochrome P450cam (CYP101) that catalyzes hydroxylation of camphor consuming two electrons and molecular oxygen per reaction cycle.Pdr has a molecular mass of 45.6 kDa and contains one tightly bound FAD per single polypeptide. Redox measurements, where sodium dithionite was used as a reductant, demonstrated that the midpoint potential of Pdr is Ϫ285 mV (2). The reduction of Pdr by NADH is a stoichiometric process that results in the formation of a long-wavelength charge transfer complex without the appearance of a semiquinone intermediate (3). The reduction of Pdr with NADPH is approximately 3 orders of magnitude slower than with NADH (4). No charge transfer band could be detected when the flavoprotein was reduced with either NADPH or sodium dithionite. Using NADPH as a reductant, the midpoint potential of Pdr was calculated to be Ϫ315 mV (4). Titration of Pdr with NADPH in the presence of excess NAD ϩ resulted in a stoichiometric reduction. This was explained as ...

Genome Mining in Sorangium cellulosum So ce56

“…1c) are typical for ferredoxin reductases with local maxima at 369 and 448 nm as well as a pronounced shoulder at 473 nm. These values correspond very well with those reported for ferredoxin reductases employed in cytochrome P450 systems such as adrenodoxin reductase (maxima 376 and 450 nm, shoulder at 475 nm (37)) or putidaredoxin reductase (maxima at 379 and 455 nm, shoulder at 480 nm (38)). In the case of FdR_B, we observed a slight bathochromic shift in the spectrum; it displayed local maxima at 388 and 457 nm as well as a shoulder at 490 nm ( (35)).…”

The Putidaredoxin Reductase-Putidaredoxin Electron Transfer Complex

“…The extinction coefficient of Pdx determined spectrophotometrically for the purest protein fractions was estimated to be 5.9 Ϯ 0.3 mM Ϫ1 cm Ϫ1 at 455 nm. Expression and purification of the 6-histidine-tagged Pdr was carried out as previously described with ⑀ 454 nm ϭ 10.9 mM Ϫ1 cm Ϫ1 used for protein concentration calculations (2).…”

“…Two molecules of Pdx, in turn, donate electrons to one molecule of P450cam that oxidizes D-camphor to 5-exo-hydroxycamphor using molecular oxygen (1). Reactions of NADH oxidation/Pdx reduction and Pdx oxidation/camphor hydroxylation are highly coupled and proceed with turnover numbers of 16,000 and 2,000 min Ϫ1 , respectively (2,3). To provide efficient catalytic turnover of P450cam monooxygenase, Pdx must form productive transient or long lived electron transfer complexes with its redox partners.…”

Crystal Structure of the Putidaredoxin Reductase·Putidaredoxin Electron Transfer Complex

“…Complex-Protein purification and preparation of the EDClinked Pdr⅐Pdx C73S/C85S complex were carried out as reported previously (6,8,18). The fusion protein was crystallized at room temperature by a hanging-drop vapor diffusion method.…”