3OJX

Disulfide crosslinked cytochrome P450 reductase inactive

Summary for 3OJX

• Entry DOI: 10.2210/pdb3ojx/pdb
• Related: 3OJW
• Descriptor: NADPH-Cytochrome P450 Reductase, FLAVIN MONONUCLEOTIDE, FLAVIN-ADENINE DINUCLEOTIDE, ... (5 entities in total)
• Functional Keywords: cytochrome p450 reductase, cypor, disulfide crosslinked, inactive, oxidoreductase
• Biological source: Rattus norvegicus (rat)
• Cellular location: Endoplasmic reticulum membrane ; Single-pass membrane protein ; Cytoplasmic side : P00388
• Total number of polymer chains: 1
• Total formula weight: 72509.49

Authors

Xia, C.,Hamdane, D.,Shen, A.,Choi, V.,Kasper, C.,Zhang, H.,Im, S.-C.,Waskell, L.,Kim, J.-J.P. (deposition date: 2010-08-23, release date: 2011-02-23, Last modification date: 2023-09-06)

Primary citation

Xia, C.,Hamdane, D.,Shen, A.L.,Choi, V.,Kasper, C.B.,Pearl, N.M.,Zhang, H.,Im, S.C.,Waskell, L.,Kim, J.J.
Conformational Changes of NADPH-Cytochrome P450 Oxidoreductase Are Essential for Catalysis and Cofactor Binding.
J.Biol.Chem., 286:16246-16260, 2011

PubMed Abstract: The crystal structure of NADPH-cytochrome P450 reductase (CYPOR) implies that a large domain movement is essential for electron transfer from NADPH via FAD and FMN to its redox partners. To test this hypothesis, a disulfide bond was engineered between residues Asp(147) and Arg(514) in the FMN and FAD domains, respectively. The cross-linked form of this mutant protein, designated 147CC514, exhibited a significant decrease in the rate of interflavin electron transfer and large (≥90%) decreases in rates of electron transfer to its redox partners, cytochrome c and cytochrome P450 2B4. Reduction of the disulfide bond restored the ability of the mutant to reduce its redox partners, demonstrating that a conformational change is essential for CYPOR function. The crystal structures of the mutant without and with NADP(+) revealed that the two flavin domains are joined by a disulfide linkage and that the relative orientations of the two flavin rings are twisted ∼20° compared with the wild type, decreasing the surface contact area between the two flavin rings. Comparison of the structures without and with NADP(+) shows movement of the Gly(631)-Asn(635) loop. In the NADP(+)-free structure, the loop adopts a conformation that sterically hinders NADP(H) binding. The structure with NADP(+) shows movement of the Gly(631)-Asn(635) loop to a position that permits NADP(H) binding. Furthermore, comparison of these mutant and wild type structures strongly suggests that the Gly(631)-Asn(635) loop movement controls NADPH binding and NADP(+) release; this loop movement in turn facilitates the flavin domain movement, allowing electron transfer from FMN to the CYPOR redox partners.

PubMed: 21345800
DOI: 10.1074/jbc.M111.230532

Experimental method

X-RAY DIFFRACTION (2.5 Å)