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Structural basis for the electron transfer from an open form of NADPH-cytochrome P450 oxidoreductase to heme oxygenase.

ABSTRACT: NADPH-cytochrome P450 oxidoreductase (CPR) supplies electrons to various heme proteins including heme oxygenase (HO), which is a key enzyme for heme degradation. Electrons from NADPH flow first to flavin adenine dinucleotide, then to flavin mononucleotide (FMN), and finally to heme in the redox partner. For electron transfer from CPR to its redox partner, the ''closed-open transition'' of CPR is indispensable. Here, we demonstrate that a hinge-shortened CPR variant, which favors an open conformation, makes a stable complex with heme-HO-1 and can support the HO reaction, although its efficiency is extremely limited. Furthermore, we determined the crystal structure of the CPR variant in complex with heme-HO-1 at 4.3-Å resolution. The crystal structure of a complex of CPR and its redox partner was previously unidentified. The distance between heme and FMN in this complex (6 Å) implies direct electron transfer from FMN to heme.

SUBMITTER: Sugishima M

PROVIDER: S-EPMC3932878 | biostudies-literature | 2014 Feb

REPOSITORIES: biostudies-literature

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Structural basis for the electron transfer from an open form of NADPH-cytochrome P450 oxidoreductase to heme oxygenase.

Sugishima Masakazu M Sato Hideaki H Higashimoto Yuichiro Y Harada Jiro J Wada Kei K Fukuyama Keiichi K Noguchi Masato M

Proceedings of the National Academy of Sciences of the United States of America 20140203 7

NADPH-cytochrome P450 oxidoreductase (CPR) supplies electrons to various heme proteins including heme oxygenase (HO), which is a key enzyme for heme degradation. Electrons from NADPH flow first to flavin adenine dinucleotide, then to flavin mononucleotide (FMN), and finally to heme in the redox partner. For electron transfer from CPR to its redox partner, the ''closed-open transition'' of CPR is indispensable. Here, we demonstrate that a hinge-shortened CPR variant, which favors an open conforma ...[more]

PMID: 24550278

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Project description:The influence of the side chains and positioning of the carboxy-terminal residues of NADPH-cytochrome P450 oxidoreductase (CYPOR) on catalytic activity, structure of the carboxy terminus, and interaction with cofactors has been investigated. A tandem deletion of residues Asp675 and Val676, that was expected to shift the position of the functionally important Trp677, resulted in higher cytochrome c reductase activity than that expected from previous studies on the importance of Asp675 and Trp677 in catalysis. Crystallographic determination of the structure of this variant revealed two conformations of the carboxy terminus. In one conformation (Mol A), the last α-helix is partially unwound, resulting in repositioning of all subsequent residues in β-strand 21, from Arg671 to Leu674 (corresponding to Ser673 and Val676 in the wild type structure). This results in the two C-terminal residues, Trp677 and Ser678, being maintained in their wild type positions, with the indole ring of Trp677 stacked against the isoalloxazine ring of FAD as seen in the wild type structure, and Ser673 occupying a similar position to the catalytic residue, Asp675. The other, more disordered conformation is a mixture of the Mol A conformation and one in which the last α-helix is not unwound and the nicotinamide ring is in one of two conformations, out towards the protein surface as observed in the wild type structure (1AMO), or stacked against the flavin ring, similar to that seen in the W677X structure that lacks Trp677 and Ser678 (1JA0). Further kinetic analysis on additional variants showed deletion or substitution of alanine or glycine for Trp677 in conjunction with deletion of Ser678 produced alterations in interactions of CYPOR with NADP+, 2'5'-ADP, and 2'-AMP, as well as the pH dependence of cytochrome c reductase activity. We postulate that deletion of bulky residues at the carboxy terminus permits increased mobility leading to decreased affinity for the 2'5'-ADP and 2'-AMP moieties of NADP+ and subsequent domain movement.

Dataset Information

Structural basis for the electron transfer from an open form of NADPH-cytochrome P450 oxidoreductase to heme oxygenase.

Publications

Structural basis for the electron transfer from an open form of NADPH-cytochrome P450 oxidoreductase to heme oxygenase.

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