Dataset Information

Probing the role of the proximal heme ligand in cytochrome P450cam by recombinant incorporation of selenocysteine.

ABSTRACT: The unique monooxygenase activity of cytochrome P450cam has been attributed to coordination of a cysteine thiolate to the heme cofactor. To investigate this interaction, we replaced cysteine with the more electron-donating selenocysteine. Good yields of the selenoenzyme were obtained by bacterial expression of an engineered gene containing the requisite UGA codon for selenocysteine and a simplified yet functional selenocysteine insertion sequence (SECIS). The sulfur-to-selenium substitution subtly modulates the structural, electronic, and catalytic properties of the enzyme. Catalytic activity decreases only 2-fold, whereas substrate oxidation becomes partially uncoupled from electron transfer, implying a more complex role for the axial ligand than generally assumed.

SUBMITTER: Aldag C

PROVIDER: S-EPMC2657087 | biostudies-literature | 2009 Apr

REPOSITORIES: biostudies-literature

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Probing the role of the proximal heme ligand in cytochrome P450cam by recombinant incorporation of selenocysteine.

Aldag Caroline C Gromov Igor A IA García-Rubio Inés I von Koenig Konstanze K Schlichting Ilme I Jaun Bernhard B Hilvert Donald D

Proceedings of the National Academy of Sciences of the United States of America 20090317 14

The unique monooxygenase activity of cytochrome P450cam has been attributed to coordination of a cysteine thiolate to the heme cofactor. To investigate this interaction, we replaced cysteine with the more electron-donating selenocysteine. Good yields of the selenoenzyme were obtained by bacterial expression of an engineered gene containing the requisite UGA codon for selenocysteine and a simplified yet functional selenocysteine insertion sequence (SECIS). The sulfur-to-selenium substitution subt ...[more]

PMID: 19293375

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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.

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.

Dataset Information

Probing the role of the proximal heme ligand in cytochrome P450cam by recombinant incorporation of selenocysteine.

Publications

Probing the role of the proximal heme ligand in cytochrome P450cam by recombinant incorporation of selenocysteine.

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