A Refined Model for the Solution Structure of Oxidized Putidaredoxin ,

Interaction and electron transfer between putidaredoxin reductase (Pdr) and putidaredoxin (Pdx) from Pseudomonas putida was studied by molecular modeling, mutagenesis, and stopped flow techniques. Based on the crystal structures of Pdr and Pdx, a complex between the proteins was generated using computer graphics methods. In the model, Pdx is docked above the isoalloxazine ring of FAD of Pdr with the distance between the flavin and In the three-component camphor hydroxylase system from Pseudomonas putida, a FAD-containing NADH-putidaredoxin reductase (Pdr) 1 receives two electrons as a hydride from NADH and delivers these as single reducing equivalents to two molecules of a [2Fe-2S] ferredoxin, putidaredoxin (Pdx). 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. To date, there is more supporting evidence for the electron transfer shuttle mechanism in the P450cam monooxygenase, according to which Pdx acts as a freely diffusible shuttle between Pdr and P450cam (4 -7), as opposed to the mechanism that requires formation of a ternary complex between the three redox partners (8, 9).Determination of the x-ray and NMR structures of P450cam (10) and Pdx (11), respectively, has enabled a better understanding structure-function relations in these proteins. The lack of structural information on Pdr, however, was the primary reason that the Pdr-Pdx redox couple was the least studied in this system. The reported data are contradictory and suggest that steric, electrostatic, or hydrophobic components are involved in the association between the flavo-and iron-sulfur proteins (5,(12)(13)(14)(15). Investigation of the Pdr-Pdx interaction is necessary for unraveling the mechanism of the P450cam and other homologous three component monooxygenase systems. The long range interprotein electron transfer is a fundamental process, and, thus, elucidation of specific interactions that assist and stabilize PdrPdx complex and mediate FAD-to-[2Fe-2S] electron transfer is of fundamental importance.Recently determined x-ray structures of oxidized Pdr and oxidized and reduced Pdx have provided a structural base for studying electron transfer and molecular recognition in the Pdr-Pdx redox couple (16 -18). In this study, we utilized computer graphics techniques and crystal structures of the proteins to develop three-dimensional models for the Pdr-Pdx complex. To test the validity of the proposed models and to further investigate structure-function relations in Pdx, four residues of the iron-sulfur protein located at the protein-protein interface in the model complexes were mutated, and the redox prop...

Section: Rationale For the Mutation Of Specific Amino Acids Ofmentioning

confidence: 99%

The Putidaredoxin Reductase-Putidaredoxin Electron Transfer Complex

Kuznetsov¹,

Blair²,

Farmer³

et al. 2005

“…In hepatic P450 systems, the redox partner is the NADPH-dependent diflavin-cytochrome P450 reductase, which contains FAD and FMN cofactors (1,5). In mammalian adrenal systems and many bacterial P450 enzymes, electrons are delivered via a two-protein redox system comprising an FAD-containing reductase (adrenodoxin reductase or ferredoxin reductase) and an iron-sulfur protein (ferredoxin) (6,7). However, other forms of redox systems supporting P450 catalysis are known to exist, including the direct interaction of P450 with hydrogen peroxide to facilitate fatty acid hydroxylation in the Bacillus subtilis P450 BS␤ enzyme (8).…”

mentioning

confidence: 99%

Flavocytochrome P450 BM3 Mutant A264E Undergoes Substrate-dependent Formation of a Novel Heme Iron Ligand Set

Girvan

Marshall

Lawson

et al. 2004

101

A conserved glutamate covalently attaches the heme to the protein backbone of eukaryotic CYP4 P450 enzymes. In the related Bacillus megaterium P450 BM3, the corresponding residue is Ala 264 . The A264E mutant was generated and characterized by kinetic and spectroscopic methods. A264E has an altered absorption spectrum compared with the wild-type enzyme (Soret maximum at ϳ420.5 nm). Fatty acid substrates produced an inhibitor-like spectral change, with the Soret band shifting to 426 nm. Optical titrations with long-chain fatty acids indicated higher affinity for A264E over the wild-type enzyme. The heme iron midpoint reduction potential in substrate-free A264E is more positive than that in wild-type P450 BM3 and was not changed upon substrate binding. EPR, resonance Raman, and magnetic CD spectroscopies indicated that A264E remains in the low-spin state upon substrate binding, unlike wild-type P450 BM3. EPR spectroscopy showed two major species in substrate-free A264E. The first has normal Cys-aqua iron ligation. The second resembles formateligated P450cam. Saturation with fatty acid increased the population of the latter species, suggesting that substrate forces on the glutamate to promote a Cys-Glu ligand set, present in lower amounts in the substratefree enzyme. A novel charge-transfer transition in the near-infrared magnetic CD spectrum provides a spectroscopic signature characteristic of the new A264E heme iron ligation state. A264E retains oxygenase activity, despite glutamate coordination of the iron, indicating that structural rearrangements occur following heme iron reduction to allow dioxygen binding. Glutamate coordination of the heme iron is confirmed by structural studies of the A264E mutant (Joyce, M. G.,

“…The ET from NADH to P450cam is sequentially mediated by a flavin group of putidaredoxin reductase and a [2Fe-2S] center of putidaredoxin (Pdx). With the availability of the P450cam (3) and Pdx (4,5) structures, many investigators have performed the kinetic (6 -9), mutational (10 -14), and theoretical (15,16) studies to clarify the molecular mechanism for the ET reaction in the P450cam⅐Pdx system. These studies demonstrated that Pdx interacts with the proximal surface of P450cam through the electrostatic interaction (12,15,17) and suggested that Arg-112 at the putative Pdx binding site forms the ET pathway in the P450cam⅐Pdx complex (12,16).…”

mentioning

confidence: 99%

“…Low potential ironsulfur protein such as spinach ferredoxin and bovine adrenodoxin can donate an electron to ferric P450cam (the first ET) but yield no hydroxylation products. On the other hand, the addition of rat liver cytochrome b 5 (cyt b 5 ) and bacterial rubredoxins to oxy-P450cam can yield the hydroxylation product, whereas these non-physiological electron donors cannot transfer the first electron to ferric P450cam. Thus, the specific complex formation between P450cam and Pdx is required for the turnover reaction.…”

mentioning

confidence: 99%

NMR Study on the Structural Changes of Cytochrome P450cam upon the Complex Formation with Putidaredoxin

Tosha

Shiro

Takahashi

et al. 2003

We investigated putidaredoxin-induced structural changes in carbonmonoxy P450cam by using NMR spectroscopy. The resonance from the ␤-proton of the axial cysteine was upfield shifted by 0.12 ppm upon the putidaredoxin binding, indicating that the axial cysteine approaches to the heme-iron by about 0.1 Å. The approach of the axial cysteine to the heme-iron would enhance the electronic donation from the axial thiolate to the heme-iron, resulting in the enhanced heterolysis of the dioxygen bond. In addition to the structural perturbation on the axial ligand, the structural changes in the substrate and ligand binding site were observed. The resonances from the 5-exo-and 9-methyl-protons of d-camphor, which were newly identified in this study, were upfield shifted by 1.28 and 0.20 ppm, respectively, implying that d-camphor moves to the heme-iron by 0.15-0.7 Å. Based on the radical rebound mechanism, the approach of d-camphor to the heme-iron could promote the oxygen transfer reaction. On the other hand, the downfield shift of the resonance from the ␥-methyl group of Thr-252 reflects the movement of the side chain away from the heme-iron by ϳ0.25 Å. Because Thr-252 regulates the heterolysis of the dioxygen bond, the positional rearrangement of Thr-252 might assist the scission of the dioxygen bond. We, therefore, conclude that putidaredoxin induces the specific heme environmental changes of P450cam, which would facilitate the oxygen activation and the oxygen transfer reaction.Cytochrome P450 (P450) 1 is a heme-containing monooxygenase, which catalyzes the hydroxylation reactions of a wide variety of natural and unnatural substrates such as steroids, fatty acids, hydrocarbons, and xenobiotics (1). The P450 catalysis reaction requires two electrons from NADH or NADPH through the redox-linked proteins. In microsomal P450s, the electron transfer (ET) from NADPH to P450s is mediated by NADPH-P450 reductase containing FMN and FAD as the redox centers. In contrast, the electron from NADH or NADPH is sequentially transferred to ferredoxin reductase, ferredoxin, and finally P450 in mitochondrial and bacterial systems. The ET reaction between P450 and its redox partner is essential for the subsequent activation of molecular oxygen and the monooxygenation reaction.To understand the mechanism of the ET reaction in P450 and its redox partner system, the ET reaction between cytochrome P450cam (P450cam) from Pseudomonas putida, one of the bacterial P450s, and its redox partner, putidaredoxin (Pdx), has intensively been investigated. P450cam catalyzes the regio-and stereo-specific hydroxylation of its substrate, d-camphor (1, 2) by accepting two electrons from NADH. The ET from NADH to P450cam is sequentially mediated by a flavin group of putidaredoxin reductase and a [2Fe-2S] center of putidaredoxin (Pdx). With the availability of the P450cam (3) and Pdx (4, 5) structures, many investigators have performed the kinetic (6 -9), mutational (10 -14), and theoretical (15, 16) studies to clarify the molecular mechanism for the ET reaction ...