Complex Formation of Cytochrome P450cam with Putidaredoxin

The cytochrome P450cam active site is known to be perturbed by binding to its redox partner, putidaredoxin (Pdx). Pdx binding also enhances the camphor monooxygenation reaction (Nagano, S., Shimada, H., Tarumi Comparison of these structures shows that the L358P mutation results in the movement of Arg-112, a residue known to be important for putidaredoxin binding, toward the heme. This change could optimize the Pdxbinding site leading to a higher affinity for Pdx. The mutation also pushes the heme toward the substrate and ligand binding pocket, which relocates the substrate to a position favorable for regio-selective hydroxylation. The camphor is held more firmly in place as indicated by a lower average temperature factor. Residues involved in the catalytically important proton shuttle system in the I helix are also altered by the mutation. Such conformational alterations and the enhanced reactivity of the mutant oxy complex with nonphysiological electron donors suggest that Pdx binding optimizes the distal pocket for monooxygenation of camphor.Cytochrome P450cam (P450cam) 1 (1, 2) catalyzes the regioand stereo-specific hydroxylation of D-camphor. To activate molecular oxygen, P450cam requires two protons and two electrons. Protons are provided from bulk water through a proton shuttle system that includes Asp-251, Thr-252, and water molecules (3-6). Electrons are provided by an iron-sulfur protein, putidaredoxin (Pdx). In the electron transfer steps, P450cam forms a complex with reduced Pdx, and the P450cam active site is perturbed as indicated by various spectroscopic studies including NMR (7, 8), EPR (9, 10), resonance Raman (11-13), and infrared (14, 15). Because nonphysiological redox proteins such as ferredoxin and adrenodoxin cannot support the monooxygenation reaction or induce the conformational changes (7, 16), the Pdxinduced changes in P450cam are thought to be important for the "effector" function of Pdx in addition to its reductase activity. In support of this view, a recent infrared and mutational study (15) has shown that Pdx-induced conformational changes promote the camphor monooxygenation reaction.Recent NMR studies found that the CO complex of the L358P mutant has very similar spectroscopic property to the WT P450cam-CO in a complex with Pdx (8,17). This suggests that the L358P mutant is a good model for Pdx-bound P450cam. However, our knowledge on the conformational change from the NMR studies is limited to only two residues, the axial cysteine ligand (Cys-357) and Thr-252 and the substrate. To overcome this limitation and to complement the NMR work, we have solved the crystal structures of the ferrous and ferrous-CO complex of the L358P mutant. EXPERIMENTAL PROCEDURESProtein Purification and Crystallization-WT P450cam and the L358P 2 mutant were overexpressed in Escherichia coli and purified as described elsewhere (18). The CO-bound P450cam structure was first solved by Raag and Poulos (19) by using the initial P2 1 2 1 2 1 crystal form used to solve the P450cam structure. For a more accurate...

Section: Resultsmentioning

confidence: 78%

mentioning

confidence: 99%

Crystal Structure of the Cytochrome P450cam Mutant That Exhibits the Same Spectral Perturbations Induced by Putidaredoxin Binding

Nagano

Tosha

Ishimori

et al. 2004

“…The tilt of the heme plane upon the binding of Pdx is supported by the shifts of the low frequency Raman lines in the 300 -800 cm Ϫ1 region (23). Although the specific assignments have not yet been reported for P450cam, the low frequency Raman lines would involve the vibrational modes of the peripheral groups of the heme as observed for myoglobin (55).…”

Section: Nmr Spectra In the Presencementioning

confidence: 70%

“…Other important feature of the P450cam⅐Pdx complex is that Pdx induces conformational changes of P450cam upon the complex formation (21)(22)(23)(24)(25)(26)(27)(28). EPR and resonance Raman studies clearly exhibited that the complex formation with Pdx converts the spin-state of ferric P450cam from the high to low spin state (21,24).…”

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 ...

“…In resonance Raman (17, 18), IR (19), NMR (20,21), and EPR studies (11,22), Pdx binding has been shown to cause protein conformational changes that distort the P450-CAM heme structure and, especially, the Fe-S bond. In particular, Tosha et al (20) proposed that Pdx binding leads to structural changes that facilitate oxygen activation, and Unno et al (18) suggested that the structural changes may correlate with H-bonding to the proximal Cys. Similarly, Nagano et al (19) proposed that Pdx binding promotes electron donation from the proximal Cys to the iron-bound O 2 to facilitate O-O bond cleavage.…”

mentioning

confidence: 99%

Direct Observation of a Novel Perturbed Oxyferrous Catalytic Intermediate during Reduced Putidaredoxin-initiated Turnover of Cytochrome P-450-CAM

Glascock

Ballou

Dawson

2005

The single turnover of (1R)(؉)-camphor-bound oxyferrous cytochrome P450-CAM with one equivalent of dithionite-reduced putidaredoxin (Pdx) was monitored for the appearance of transient intermediates at 3°C by double mixing rapid scanning stopped-flow spectroscopy. With excess camphor, three successive species were observed after generating oxyferrous P450-CAM and reacting versus reduced Pdx: a perturbed oxyferrous derivative, a species that was a mixture of high and low spin Fe(III), and high spin ferric camphor-bound enzyme. The rates of the first two steps, ϳ140 and ϳ85 s ؊1, were assigned to formation of the perturbed oxyferrous intermediate and to electron transfer from reduced Pdx, respectively. In the presence of stoichiometric substrate, three phases with similar rates were seen even though the final state is low spin ferric P450-CAM. This is consistent with substrate being hydroxylated during the reaction. The single turnover reaction initiated by adding dioxygen to a preformed reduced P450-CAM⅐Pdx complex with excess camphor also led to phases with similar rates. It is proposed that formation of the perturbed oxyferrous intermediate reflects alteration of H-bonding to the proximal Cys, increasing the reduction potential of the oxyferrous state and triggering electron transfer from reduced Pdx. This species may be a direct spectral signature of the effector role of Pdx on P450-CAM reactivity (i.e. during catalysis). The substrate-free oxyferrous enzyme also reacted readily with reduced Pdx, showing that the inability of substratefree P450-CAM to accept electrons from reduced Pdx and function as an NADH oxidase is completely due to the incapacity of reduced Pdx to deliver the first but not the second electron.The cytochrome P-450 family of heme-containing mono-oxygenases is involved in the metabolism of xenobiotics and in the production of physiologically important molecules (1). A defining characteristic of the P-450 family is the proximal thiolate-ligated heme. P450-CAM (P450-CAM, CYP101) 3 from Pseudomonas putida catalyzes the hydroxylation of (1R)(ϩ)-camphor to form (1R)(ϩ)-5-exo-hydroxycamphor (Reaction 1). The electrons required for the reaction flow from NADH to the flavoprotein, putidaredoxin reductase, then to the iron-sulfur (2Fe/2S) protein, putidaredoxin (Pdx), and finally to P450-CAM. In the widely quoted P450 reaction cycle shown in Fig. 1 (1), substrate binding converts the ferric low spin resting state (1) to the ferric high spin form (2). Electron transfer from reduced Pdx gives high spin deoxyferrous P-450 (3). Dioxygen binding yields the oxy-ferrous adduct, a ferrous-O 2 /ferric superoxide resonance hybrid (4a 7 4b). CO binding to 3 generates the ferrous-CO derivative (5). Addition of the second electron from reduced Pdx has been proposed to yield a ferric peroxo species (6a), protonation of which gives the hydroperoxo state (6b). Protonation of the distal oxygen produces water and compound I, a ferryl porphyrin -cation radical (7). This highly oxidizing intermediate abstracts a hydrogen...