Probing the Interactions of Putidaredoxin with Redox Partners in Camphor P450 5-Monooxygenase by Mutagenesis of Surface Residues

Holden, Marcia J.; Mayhew, Martin P.; Bunk, David M.; Roitberg, Adrián E.; Vilker, Vincent L.

doi:10.1074/jbc.272.35.21720

Cited by 79 publications

(111 citation statements)

References 29 publications

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“…However, the crystal and solution structures of the ET complex formed by the two molecules were solved only very recently by X-ray crystallography and paramagnetic NMR (23, 24). The relative orientation of the proteins within the complex in the solution and crystalline states is practically identical, consistent with the information available from the mutagenesis studies of the system and favorable for effective ET (21,[25][26][27][28][29]. It is still under debate whether Pdx induces opening of substrate-bound cytP450cam.…”

supporting

confidence: 61%

Identification of productive and futile encounters in an electron transfer protein complex

Andrałojć

Hiruma

Wei-min

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

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Well-defined, stereospecific states in protein complexes are often in exchange with an ensemble of more dynamic orientations: the encounter states. The structure of the stereospecific complex between cytochrome P450cam and putidaredoxin was solved recently by X-ray diffraction as well as paramagnetic NMR spectroscopy. Other than the stereospecific complex, the NMR data clearly show the presence of additional states in the complex in solution. In these encounter states, populated for a small percentage of the time, putidaredoxin assumes multiple orientations and samples a large part of the surface of cytochrome P450cam. To characterize the nature of the encounter states, an extensive paramagnetic NMR dataset has been analyzed using the Maximum Occurrence of Regions methodology. The analysis reveals the location and maximal spatial extent of the additional states needed to fully explain the NMR data. Under the assumption of sparsity of the size of the conformational ensemble, several minor states can be located quite precisely. The distribution of these minor states correlates with the electrostatic potential map around cytochrome P450cam. Whereas some minor states are on isolated positively charged patches, others are connected to the stereospecific site via positively charged paths. The existence of electrostatically favorable pathways between the stereospecific interaction site and the different minor states or lack thereof suggests a means to discriminate between productive and futile encounter states.paramagnetic NMR | encounter complex | cytochrome P450cam | putidaredoxin | maximum occurrence C rystal structures suggest that proteins assume unique, stereospecific orientations within protein-protein complexes. However, a number of studies in solution have made clear that encounter states are an inherent element of protein complexes (1-8), especially in electron transfer (ET), where the interactions are often extremely fast (9). In the encounter complex, the proteins assume multiple other orientations, often in equilibrium with the major stereospecific state. In low-affinity complexes with dissociation constant (K d ) values > 10 μM, the encounter complex can represent a sizeable fraction, and in some cases, a well-defined, stereospecific complex may even be absent (10-15). The presence of encounter states may be a consequence of the chemical nature of proteins. In nonobligate stereospecific complexes, the interface represents a small fraction of the total protein surface, and therefore, it is reasonable to assume that weak interactions also occur elsewhere. In the case in which protein pairs have evolved to exhibit a high association rate by using electrostatic preorientation, electrostatic patches seem to enhance the presence of encounter states (16,17). On the one hand, the preorientation reduces the surface area that is visited by the partner, thus enhancing the number of productive encounters, but on the other hand, highly charged patches can bind the oppositely charged protein in many orientations with a...

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supporting

confidence: 61%

Identification of productive and futile encounters in an electron transfer protein complex

Andrałojć

Hiruma

Wei-min

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

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“…Dual Function of Pdr-Cysteine 73 of Pdx has been identified as important in the interaction with Pdr, since its replacement with other amino acid residues resulted in more than 60% loss of activity toward the flavoprotein (12). One of the findings in this study was the lack of effect of the sulfhydryl group modification of Pdr on its Pdx reductase activity.…”

Section: Discussionmentioning

confidence: 51%

“…A low rate conversion of DTNB to TNB occurred in the absence of enzyme, and this was subtracted from the rate in the presence of enzyme. Pdx reduction activity was measured at 455 nm using an extinction coefficient of 2.5 mM Ϫ1 cm Ϫ1 as previously described (12). Specific reductase activities were calculated employing molar extinction coefficients of 6.22 mM Ϫ1 cm Ϫ1 at 340 nm, 1.02 mM Ϫ1 cm Ϫ1 at 420 nm, and 21 mM Ϫ1 cm Ϫ1 at 600 nm for NADH, ferricyanide, and DCIP, respectively.…”

Section: Methodsmentioning

confidence: 99%

Putidaredoxin Reductase, a New Function for an Old Protein

Sevrioukova¹,

Poulos²

2002

Journal of Biological Chemistry

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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 ...

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“…To correlate the spectral changes with the ET activity, we followed the spectral changes by addition of a Pdx mutant, Asp-38 3 Asn (D38N), whose ET activity is nearly abolished (13,14). Fig.…”

Section: Nmr Spectral Changes Upon the Binding Of The Pdx Mutant-prevmentioning

confidence: 99%

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

Tosha

Shiro

Takahashi

et al. 2003

Journal of Biological Chemistry

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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 ...

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Probing the Interactions of Putidaredoxin with Redox Partners in Camphor P450 5-Monooxygenase by Mutagenesis of Surface Residues

Cited by 79 publications

References 29 publications

Identification of productive and futile encounters in an electron transfer protein complex

Identification of productive and futile encounters in an electron transfer protein complex

Putidaredoxin Reductase, a New Function for an Old Protein

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

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