Specific effects of potassium ion binding on wild-type and L358P cytochrome P450cam.
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.
OuYang
Bo
B
Department of Chemistry, Brandeis University, 415 South Street, MS 015, Waltham, Massachusetts 02454-9110, USA.
Pochapsky
Susan Sondej
SS
Pagani
Gina M
GM
Pochapsky
Thomas C
TC
D019475:Camphor 5-Monooxygenase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D002412:Cations / Q000737:chemistry; D008239:Lysine / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D009154:Mutation / Q000235:genetics; D019906:Nuclear Magnetic Resonance, Biomolecular; D010084:Oxidation-Reduction; D010649:Phenylalanine / Q000235:genetics / Q000378:metabolism*; D011188:Potassium / Q000737:chemistry* / Q000378:metabolism*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D013455:Sulfur / Q000737:chemistry / Q000378:metabolism
D002412:Cations; D010649:Phenylalanine; D013455:Sulfur; D019475:Camphor 5-Monooxygenase; D008239:Lysine; D011188:Potassium