Specific Effects of Potassium Ion Binding on Wild-Type and L358P Cytochrome P450cam

Abstract

The camphor monoxygenase cytochrome P450 cam (CYP101) requires potassium ion (K + ) to drive formation of the characteristic high-spin state of the heme Fe +3 upon substrate binding. Amide 1 H, 15 N correlations in perdeuterated [U-15 N] 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 Pdx r titration. However, Pdx r 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.

The Metal Drives the Chemistry: Dual Functions of Acireductone Dioxygenase

“…Although many metal-containing co-factors (e.g., heme, chlorophyll, cobalamin) are pre-formed when finally bound to their protein partners, this still requires that the co-factors themselves be properly synthesized with the correct metal. Alkali metal and alkaline earth ions are sometimes bound via amide carbonyl oxygens (as in selective ion channels 110 or when stabilizing local structural motifs 111 ). In these cases, the protein backbone conformations appear to be pre-arranged to selectively bind cations of the appropriate size and charge.…”

Reversible inactivation of cytochrome P450 by alkaline earth metal ions: Auxiliary metal ion induced conformation change and formation of inactive P420 species in CYP101

“…Binding of potassium was proposed to stabilize an otherwise unfavourable B 0 helix in the enzyme leading to a conformational change resulting in the removal of water from the active site and an increase in the affinity for the substrate [8,[11][12][13]. A recent NMR study demonstrated widespread change in the backbone polypeptide conformation of the enzyme as a result of potassium binding to the enzyme in presence of camphor [14]. Substitution of the potassium ion by monovalent cations were shown to drastically decrease the camphor binding affinity of P450cam [8].…”

Delicate conformational balance of the redox enzyme cytochrome P450cam

“…Thus, we conclude that upon binding of Pdx, oxidized P450cam remains in the closed state in solution at 25°C. All studies on the structure of P450cam in the presence of Pdx used samples containing between 50 and 200 mM KCl, and potassium ions have been implicated in stabilizing the closed form of P450cam when camphor is bound (41). Therefore, in this study, the Leu-labeled samples used to determine the state of P450cam in the presence of Pdx (Table S1, (42).…”