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Crystal structure and characterization of a cytochrome c peroxidase-cytochrome c site-specific cross-link.

ABSTRACT: A specific covalently cross-linked complex between redox partners yeast cytochrome c peroxidase (CCP) and cytochrome c (cyt. c) has been made by engineering cysteines into CCP and cyt. c that form an intermolecular disulfide bond in high yield. The crystal structure of the cross-linked complex has been solved to 1.88-A resolution and closely resembles the structure of the noncovalent complex [Pellitier, H. & Kraut, J. (1992) Science 258, 1748-1755]. The higher resolution of the covalent complex has enabled the location of ordered water molecules at the peroxidase-cytochrome c interface that serve to bridge between the two proteins by hydrogen bonding. As in the noncovalent complex, direct electrostatic interactions between protein groups appear not to be critical in complex formation. UV-visible spectroscopic and stopped-flow studies indicate that CCP in the covalent complex reacts normally with H(2)O(2) to give compound I. Stopped-flow kinetic studies also show that intramolecular electron transfer between the cross-linked ferrocytochrome c and the Trp-191 cation radical site in CCP compound I occurs fast and is nearly complete within the dead time ( approximately 2 ms) of the instrument. These results indicate that the structure of the covalent complex closely mimics the physiological electron transfer complex. In addition, single-turnover and steady-state experiments reveal that CCP compound I in the covalent complex oxidizes exogenously added ferrocytochrome c at a slow rate (t(1/2) approximately 2 min), indicating that CCP does not have a second independent site for physiologically relevant electron transfer.

SUBMITTER: Guo M

PROVIDER: S-EPMC395902 | biostudies-literature | 2004 Apr

REPOSITORIES: biostudies-literature

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Crystal structure and characterization of a cytochrome c peroxidase-cytochrome c site-specific cross-link.

Guo Maolin M Bhaskar B B Li Huiying H Barrows Tiffany P TP Poulos Thomas L TL

Proceedings of the National Academy of Sciences of the United States of America 20040407 16

A specific covalently cross-linked complex between redox partners yeast cytochrome c peroxidase (CCP) and cytochrome c (cyt. c) has been made by engineering cysteines into CCP and cyt. c that form an intermolecular disulfide bond in high yield. The crystal structure of the cross-linked complex has been solved to 1.88-A resolution and closely resembles the structure of the noncovalent complex [Pellitier, H. & Kraut, J. (1992) Science 258, 1748-1755]. The higher resolution of the covalent complex ...[more]

PMID: 15071191

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Project description:DNA interstrand cross-links (ICLs) are products of chemotherapeutic agents and cellular metabolic processes that block both replication and transcription. If left unrepaired, ICLs are extremely toxic to cells, and ICL repair mechanisms contribute to the survival of certain chemotherapeutic resistance tumors. A critical step in ICL repair involves unhooking the cross-link. In the absence of a homologous donor sequence, the resulting gap can be filled in by a repair synthesis step involving bypass of the cross-link remnant. Here, we examine the effect of cross-link structure on the ability of unhooked DNA substrates to undergo repair synthesis in mammalian whole cell extracts. Using 32P incorporation assays, we found that repair synthesis occurs efficiently past the site of damage when a DNA substrate containing a single N4C-ethyl-N4C cross-link is incubated in HeLa or Chinese hamster ovary cell extracts. This lesion, which can base pair with deoxyguanosine, is readily bypassed by both Escherichia coli DNA polymerase I and T7 DNA polymerase in a primer extension assay. In contrast, bypass was not observed in the primer extension assay or in mammalian cell extracts when DNA substrates containing a N3T-ethyl-N3T or N1I-ethyl-N3T cross-link, whose linkers obstruct the hydrogen bond face of the bases, were used. A modified phosphorothioate sequencing method was used to analyze the ICL repair patches created in the mammalian cell extracts. In the case of the N4C-ethyl-N4C substrate, the repair patch spanned the site of the cross-link, and the lesion was bypassed in an error-free manner. However, although the N3T-ethyl-N3T and N1I-ethyl-N3T substrates were unhooked in the extracts, bypass was not detected. These and our previous results suggest that although the chemical structure of an ICL may not affect initial cross-link unhooking, it can play a significant role in subsequent processing of the cross-link. Understanding how the physical and chemical differences of ICLs affect repair may provide a better understanding of the cytotoxic and mutagenic potential of specific ICLs.

Dataset Information

Crystal structure and characterization of a cytochrome c peroxidase-cytochrome c site-specific cross-link.

Publications

Crystal structure and characterization of a cytochrome c peroxidase-cytochrome c site-specific cross-link.

Similar Datasets

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