Probing the role of the proximal heme ligand in cytochrome P450cam by recombinant incorporation of selenocysteine.
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ABSTRACT:
SUBMITTER: Aldag C
PROVIDER: S-EPMC2657087 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:In this work we show that ligand migration and active site conformational relaxation can occur independently of each other in hemoproteins. The complicated kinetics of carbon monoxide rebinding with cytochrome P450cam display up to five distinct processes between 77 K and 300 K. They were disentangled by using a combination of three approaches: 1), the competition of the ligand with xenon for the occupation of internal protein cavities; 2), the modulation of the amount of distal steric hindrance within the heme pocket by varying the nature of the substrate; and 3), molecular mechanics calculations to support the proposed heme-substrate relaxation mechanism and to seek internal cavities. In cytochrome P450cam, active site conformational relaxation results from the displacement of the substrate toward the heme center upon photodissociation of the ligand. It is responsible for the long, puzzling bimodal nature of the rebinding kinetics observed down to 77 K. The relaxation rate is strongly substrate-dependent. Ligand migration is slower and is observed only above 135 K. Migration and return rates are independent of the substrate.
| S-EPMC1305127 | biostudies-literature
Project description:Cytochromes P450 play key roles in drug metabolism and disease by oxidizing a wide variety of natural and xenobiotic compounds. High-resolution crystal structures of P450cam bound to ruthenium sensitizer-linked substrates reveal an open conformation of the enzyme that allows substrates to access the active center via a 22-A deep channel. Interactions of alkyl and fluorinated biphenyl linkers with the channel demonstrate the importance of exploiting protein dynamics for specific inhibitor design. Large changes in peripheral enzyme structure (F and G helices) couple to conformational changes in active center residues (I helix) implicated in proton pumping and dioxygen activation. Common conformational states among P450cam and homologous enzymes indicate that static and dynamic variability in the F/G helix region allows the 54 human P450s to oxidize thousands of substrates.
| S-EPMC60069 | biostudies-literature
Project description:Although extensive research has been performed on various cytochrome P450s, especially Cyt P450cam, there is much to be learned about the mechanism of how its functional unit, a heme b ligated by an axial cysteine, is finely tuned for catalysis by its second coordination sphere. Here we study how the hydrogen-bonding network affects the proximal cysteine and the Fe-S(Cys) bond in ferric Cyt P450cam. This is accomplished using low-temperature magnetic circular dichroism (MCD) spectroscopy on wild-type (wt) Cyt P450cam and on the mutants Q360P (pure ferric high-spin at low temperature) and L358P where the "Cys pocket" has been altered (by removing amino acids involved in the hydrogen-bonding network), and Y96W (pure ferric low-spin). The MCD spectrum of Q360P reveals fourteen electronic transitions between 15200 and 31050 cm(-1). Variable-temperature variable-field (VTVH) saturation curves were used to determine the polarizations of these electronic transitions with respect to in-plane (xy) and out-of-plane (z) polarization relative to the heme. The polarizations, oscillator strengths, and TD-DFT calculations were then used to assign the observed electronic transitions. In the lower energy region, prominent bands at 15909 and 16919 cm(-1) correspond to porphyrin (P) → Fe charge transfer (CT) transitions. The band at 17881 cm(-1) has distinct sulfur S(π) → Fe CT contributions. The Q band is observed as a pseudo A-term (derivative shape) at 18604 and 19539 cm(-1). In the case of the Soret band, the negative component of the expected pseudo A-term is split into two features due to mixing with another π → π* and potentially a P → Fe CT excited state. The resulting three features are observed at 23731, 24859, and 25618 cm(-1). Most importantly, the broad, prominent band at 28570 cm(-1) is assigned to the S(σ) → Fe CT transition, whose intensity is generated through a multitude of CT transitions with strong iron character. For wt, Q360P, and L358P, this band occurs at 28724, 28570, and 28620 cm(-1), respectively. The small shift of this feature upon altering the hydrogen bonds to the proximal cysteine indicates that the role of the Cys pocket is not primarily for electronic fine-tuning of the sulfur donor strength but is more for stabilizing the proximal thiolate against external reactants (NO, O(2), H(3)O(+)), and for properly positioning cysteine to coordinate to the iron center. This aspect is discussed in detail.
| S-EPMC3035745 | biostudies-literature
Project description:Local protein backbone dynamics of the camphor hydroxylase cytochrome P450(cam) (CYP101) depend upon the oxidation and ligation state of the heme iron. (1)H-(15)N correlation nuclear magnetic resonance experiments were used to compare backbone dynamics of oxidized and reduced forms of this 414-residue metalloenzyme via hydrogen-deuterium exchange kinetics (H-D exchange) and (15)N relaxation measurements, and these results are compared with previously published results obtained by H-D exchange mass spectrometry. In general, the reduced enzyme exhibits lower-amplitude motions of secondary structural features than the oxidized enzyme on all of the time scales accessible to these experiments, and these differences are more pronounced in regions of the enzyme involved in substrate access to the active site (B' helix and beta3 and beta5 sheets) and binding of putidaredoxin (C and L helices), the iron-sulfur protein that acts as the effector and reductant of CYP101 in vivo. These results are interpreted in terms of local structural effects of changes in the heme oxidation state, and the relevance of the observed effects to the enzyme mechanism is discussed.
| S-EPMC2747118 | biostudies-literature
Project description:Cytochromes c comprise a diverse and widespread family of proteins containing covalently bound heme that are central to the life of most organisms. In many bacteria and in certain mitochondria, the synthesis of cytochromes c is performed by a complex post-translational modification apparatus called System I (or cytochrome c maturation, Ccm, system). In Escherichia coli , there are eight maturation proteins, several of which are involved in heme handling, but the mechanism of heme transfer from one protein to the next is not known. Attachment of the heme to the apocytochrome occurs via a novel covalent bond to a histidine residue of the heme chaperone CcmE. The discovery of a variant maturation system (System I*) has provided a new tool for studying cytochrome c assembly because the variant CcmE functions via a cysteine residue in the place of the histidine of System I. In this work, we use site-directed mutagenesis on both maturation systems to probe the function of the individual component proteins as well as their concerted action in transferring heme to the cytochrome c substrate. The roles of CcmA, CcmC, CcmE, and CcmF in the heme delivery process are compared between Systems I and I*. We show that a previously proposed quinone-binding site on CcmF is not essential for either system. Significant differences in the heme chemistry involved in the formation of cytochromes c in the variant system add new pieces to the cytochrome c biogenesis puzzle.
| S-EPMC3806149 | biostudies-other
Project description:The two-protein complex between putidaredoxin (Pdx) and cytochrome P450(cam) (CYP101) is the catalytically competent species for camphor hydroxylation by CYP101. We detected a conformational change in CYP101 upon binding of Pdx that reorients bound camphor appropriately for hydroxylation. Experimental evidence shows that binding of Pdx converts a single X-proline amide bond in CYP101 from trans or distorted trans to cis. Mutation of proline 89 to isoleucine yields a mixture of both bound camphor orientations, that seen in Pdx-free and that seen in Pdx-bound CYP101. A mutation in CYP101 that destabilizes the cis conformer of the Ile 88-Pro 89 amide bond results in weaker binding of Pdx. This work provides direct experimental evidence for involvement of X-proline isomerization in enzyme function.
| S-EPMC2581830 | biostudies-literature
Project description:Cytochrome c552 from Thermus thermophilus is one of the hot topics for creating smart biomaterials as it possesses remarkable stability, is tolerant to multiple mutations and has therefore been recently reported for a number of functionalizations upon substitution of the original prosthetic group with an artificial prosthetic group. However, all of the substitutions were driven by the coordination through the axial ligands followed by complete reconstitution with a metal-porphyrin complex. This limits the scope of the cytochrome c for incorporating a metal-less non-natural heme species that could improve the versatility of cytochrome c for a new generation of engineered cytochrome proteins for further enhancement in their functionalities such as biocatalysts. In this connection, a new variant of Cytochrome c (rC552 C14A) from Thermus thermophilus was reported, where an easy approach to remove the original prosthetic group was achieved, followed by the incorporation of a number of metal-PPIX derivatives that ultimately led to the formation of artificial c-type cytochromes through covalent bonding. The apo-cytochrome was found to be thermally tolerant and to possess a distinctive overall structure as that of the wild type, as was evident from the corresponding CD spectra, which ultimately encouraged reconstitution with a metal-less protoporphyrin derivative for better understanding the role of axial ligands in the reconstitution process. Successful reconstitution was achieved, resulting in a new type of Cytochrome b-type artificial protein without the metal in its active site, indicating the non-involvement of the axial ligand. In order to prove the non-involvement of the axial ligand, a subsequent double mutant (C14A/M69A) was constructed, replacing the methionine at 69 position with non-coordinating alanine residue. Accordingly, the apo-C14A/M69A was prepared and found to be extremely stable as the earlier mutants and the WT showed no signs of denaturation, even at the elevated temperature of 98°C. Subsequently, heme b was successfully incorporated into the apo-C14A/M69A, which demonstrated itself as a highly thermally tolerant protein scaffold for incorporating a metal-less artificial prosthetic group in the absence of the axial ligand. Further improvement in the reconstitution process is achieved by replacing the methionine at 69 position with phenyl alanine (C14A/M69F mutant), resulting in further stabilization of heme species, possibly through non-covalent π-interactions, as corroborated by molecular docking.
| S-EPMC10635480 | biostudies-literature
Project description:The binding of (+)-camphor to cytochrome P450cam (P450cam) expels a cluster of waters at the active site, raising the redox potential of the haem to an extent that allows reduction by the electron-transfer system. This binding was reported to involve no significant structural changes in the protein. Here, two ferric P450cam structures partially complexed with (+)-camphor were determined by X-ray crystallography at 1.30-1.35 A resolution, revealing the structures of the substrate-free and substrate-bound forms. (+)-Camphor binding induces rotation of Thr101 to form a hydrogen bond that acts as a hydrogen donor to a peripheral haem propionate. This bonding contributes to the redox-potential change.
| S-EPMC2635880 | biostudies-literature
Project description:A hydrogen bond network has been identified that adjusts protein-substrate contacts in cytochrome P450(cam) (CYP101A1). Replacing the native substrate camphor with adamantanone or norcamphor causes perturbations in NMR-detected NH correlations assigned to the network, which includes portions of a ? sheet and an adjacent helix that is remote from the active site. A mutation in this helix reduces enzyme efficiency and perturbs the extent of substrate-induced spin state changes at the haem iron that accompany substrate binding. In turn, the magnitude of the spin state changes induced by alternate substrate binding parallel the NMR-detected perturbations observed near the haem in the enzyme active site.
| S-EPMC3071453 | biostudies-literature
Project description:The formate dehydrogenases (Fdh) Fdh-O, Fdh-N, and Fdh-H, are the only proteins in Escherichia coli that incorporate selenocysteine at a specific position by decoding a UGA codon. However, an excess of selenium can lead to toxicity through misincorporation of selenocysteine into proteins. To determine whether selenocysteine substitutes for cysteine, we grew Escherichia coli in the presence of excess sodium selenite. The respiratory Fdh-N and Fdh-O enzymes, along with nitrate reductase (Nar) were co-purified from wild type strain MC4100 after anaerobic growth with nitrate and either 2 µM or 100 µM selenite. Mass spectrometric analysis of the catalytic subunits of both Fdhs identified the UGA-specified selenocysteine residue and revealed incorporation of additional, 'non-specific' selenocysteinyl residues, which always replaced particular cysteinyl residues. Although variable, their incorporation was not random and was independent of the selenite concentration used. Notably, these cysteines are likely to be non-essential for catalysis and they do not coordinate the iron-sulfur cluster. The remaining cysteinyl residues that could be identified were never substituted by selenocysteine. Selenomethionine was never observed in our analyses. Non-random substitution of particular cysteinyl residues was also noted in the electron-transferring subunit of both Fdhs as well as in the subunits of the Nar enzyme. Nar isolated from an E. coli selC mutant also showed a similar selenocysteine incorporation pattern to the wild-type indicating that non-specific selenocysteine incorporation was independent of the specific selenocysteine pathway. Thus, selenide replaces sulfide in the biosynthesis of cysteine and misacylated selenocysteyl-tRNA(Cys) decodes either UGU or UGC codons, which usually specify cysteine. Nevertheless, not every UGU or UGC codon was decoded as selenocysteine. Together, our results suggest that a degree of misincorporation of selenocysteine into enzymes through replacement of particular, non-essential cysteines, is tolerated and this might act as a buffering system to cope with excessive intracellular selenium.
| S-EPMC3636253 | biostudies-literature