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Specific effects of potassium ion binding on wild-type and L358P cytochrome P450cam.
- Source :
-
Biochemistry [Biochemistry] 2006 Dec 05; Vol. 45 (48), pp. 14379-88. - Publication Year :
- 2006
-
Abstract
- The camphor monoxygenase cytochrome P450cam (CYP101) requires potassium ion (K+) to drive formation of the characteristic high-spin state of the heme Fe+3 upon substrate binding. Amide 1H, 15N correlations in perdeuterated [U-15N] CYP101 were monitored as a function of K+ concentration by 2D-TROSY-HSQC in both camphor-bound oxidized (CYP-S) and camphor- and CO-bound reduced CYP101 (CYP-S-CO). In both forms, K+-induced spectral perturbations are detected in the vicinity of the K+ binding site proposed from crystallographic structures, but are larger and more widespread structurally in CYP-S than in CYP-S-CO. In CYP-S-CO, K+-induced perturbations occur primarily near the proposed K+ binding site in the B-B' loop and B' helix, which are also perturbed by binding of effector, putidaredoxin (Pdx). The spectral effects of K+ binding in CYP-S-CO oppose those observed upon Pdxr titration. However, Pdxr titration of CYP-S-CO in the absence of K+ results in multiple conformations. The spin-state equilibrium in the L358P mutant of CYP101 is more sensitive to K+ concentration than WT CYP101, consistent with a hypothesis that L358P preferentially populates conformations enforced by Pdx binding in WT CYP101. Thallium(I), a K+ mimic, minimizes the effects of Pdx titration on the NMR spectrum of CYP-S-CO, but is competent to replace K+ in driving the formation of high-spin CYP-S. These observations suggest that the role of K+ is to stabilize conformers of CYP-S that drive the spin-state change prior to the first electron transfer, and that K+ stabilizes the CYP-S-CO conformer that interacts with Pdx. However, upon binding of Pdx, further conformational changes occur that disfavor K+ binding.
- Subjects :
- Camphor 5-Monooxygenase genetics
Cations chemistry
Lysine genetics
Models, Molecular
Mutation genetics
Nuclear Magnetic Resonance, Biomolecular
Oxidation-Reduction
Phenylalanine genetics
Protein Binding
Protein Structure, Tertiary
Sulfur chemistry
Sulfur metabolism
Camphor 5-Monooxygenase chemistry
Camphor 5-Monooxygenase metabolism
Lysine metabolism
Phenylalanine metabolism
Potassium chemistry
Potassium metabolism
Subjects
Details
- Language :
- English
- ISSN :
- 0006-2960
- Volume :
- 45
- Issue :
- 48
- Database :
- MEDLINE
- Journal :
- Biochemistry
- Publication Type :
- Academic Journal
- Accession number :
- 17128977
- Full Text :
- https://doi.org/10.1021/bi0617355