Dataset Information

Selenolate complexes of CYP101 and the heme-bound hHO-1/H25A proximal cavity mutant.

ABSTRACT: Thiolate and selenolate complexes of CYP101 (P450cam) and the H25A proximal cavity mutant of heme-bound human heme oxygenase-1 (hHO-1) have been examined by UV-vis spectroscopy. Both thiolate and selenolate ligands bound to the heme distal side in CYP101 and gave rise to characteristic hyperporphyrin spectra. Thiolate ligands also bound to the proximal side of the heme in the cavity created by the H25A mutation in hHO-1, giving a Soret absorption similar to that of the H25C hHO-1 mutant. Selenolate ligands also bound to this cavity mutant under anaerobic conditions but reduced the heme iron to the ferrous state, as shown by the formation of a ferrous CO complex. Under aerobic conditions, the selenolate ligand but not the thiolate ligand was rapidly oxidized. These results indicate that selenocysteine-coordinated heme proteins will not be stable species in the absence of a redox potential stabilizing effect.

SUBMITTER: Jiang Y

PROVIDER: S-EPMC2727152 | biostudies-literature | 2008 May

REPOSITORIES: biostudies-literature

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Selenolate complexes of CYP101 and the heme-bound hHO-1/H25A proximal cavity mutant.

Jiang Yongying Y Ortiz de Montellano Paul R PR

Inorganic chemistry 20080401 9

Thiolate and selenolate complexes of CYP101 (P450cam) and the H25A proximal cavity mutant of heme-bound human heme oxygenase-1 (hHO-1) have been examined by UV-vis spectroscopy. Both thiolate and selenolate ligands bound to the heme distal side in CYP101 and gave rise to characteristic hyperporphyrin spectra. Thiolate ligands also bound to the proximal side of the heme in the cavity created by the H25A mutation in hHO-1, giving a Soret absorption similar to that of the H25C hHO-1 mutant. Selenol ...[more]

PMID: 18376820

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Project description:Expression of mutant EcoRII methyltransferase protein (M.EcoRII-C186A) in Escherichia coli leads to tightly bound DNA-protein complexes (TBCs), located sporadically on the chromosome rather than in tandem arrays. The mechanisms behind the lethality induced by such sporadic TBCs are not well studied, nor is it clear whether very tight binding but non-covalent complexes are processed in the same way as covalent DNA-protein crosslinks (DPCs). Using 2D gel electrophoresis, we found that TBCs induced by M.EcoRII-C186A block replication forks in vivo. Specific bubble molecules were detected as spots on the 2D gel, only when M.EcoRII-C186A was induced, and a mutation that eliminates a specific EcoRII methylation site led to disappearance of the corresponding spot. We also performed a candidate gene screen for mutants that are hypersensitive to TBCs induced by M.EcoRII-C186A. We found several gene products necessary for protection against these TBCs that are known to also protect against DPCs induced with wild-type M.EcoRII (after 5-azacytidine incorporation): RecA, RecBC, RecG, RuvABC, UvrD, FtsK, XerCD and SsrA (tmRNA). In contrast, the RecFOR pathway and Rep helicase are needed for protection against TBCs but not DPCs induced by M.EcoRII. We propose that stalled fork processing by RecFOR and RecA promotes release of tightly bound (but non-covalent) blocking proteins, perhaps by licensing Rep helicase-driven dissociation of the blocking M.EcoRII-C186A. Our studies also argued against the involvement of several proteins that might be expected to protect against TBCs. We took the opportunity to directly compare the sensitivity of all tested mutants to two quinolone antibiotics, which target bacterial type II topoisomerases and induce a unique form of DPC. We uncovered rep, ftsK and xerCD as novel quinolone hypersensitive mutants, and also obtained evidence against the involvement of a number of functions that might be expected to protect against quinolones.

Project description:Vibrational coherence spectroscopy (VCS) is used to investigate the low-frequency dynamics of camphor-free and camphor-bound cytochrome P450(cam) (CYP 101) and its L358P mutant. The low-frequency heme vibrations are found to be perturbed upon binding to the electron transfer partner putidaredoxin (Pdx). A strong correlation between the "detuned" vibrational coherence spectrum, which monitors frequencies between 100 and 400 cm(-1), and the lower frequency part of the Raman spectrum is also demonstrated. The very low frequency region ?200 cm(-1), uniquely accessed by open-band VCS measurements, reveals a mode near 103 cm(-1) in P450(cam) when camphor is not present in the distal pocket. This reflects the presence of a specific heme distortion, such as saddling or ruffling, in the substrate-free state where water is coordinated to the low-spin iron atom. Such distortions are likely to retard the rate of electron transfer to the substrate-free protein. The presence of strong mode near ?33 cm(-1) in the camphor-bound form suggests a significant heme-doming distortion, which is supported by analysis using normal coordinate structural decomposition. Pdx also displays a strong coherent vibration near 30 cm(-1) that in principle could be involved in vibrational resonance with its electron transfer target. A splitting of the 33 cm(-1) feature and intensification of a mode near 78 cm(-1) appear when the P450(cam)/Pdx complex is formed. These observations are consistent with vibrational mixing and heme geometric distortions upon Pdx binding that are coincident with the increased thiolate electron donation to the heme. The appearance of a mode near 65 cm(-1) in the coherence spectra of the L358P mutant is comparable to the mode at 78 cm(-1) seen in the P450(cam)/Pdx complex and is consistent with the view that the heme and its environment in the L358P mutant are similar to the Pdx-bound native protein. Resonance Raman spectra are presented for both P450(cam) and the L358P mutant and the changes are correlated with an increased amount of thiolate electron donation to the heme in the mutant sample.

Dataset Information

Selenolate complexes of CYP101 and the heme-bound hHO-1/H25A proximal cavity mutant.

Publications

Selenolate complexes of CYP101 and the heme-bound hHO-1/H25A proximal cavity mutant.

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