Probing the Heme Iron Coordination Structure of Pressure-Induced Cytochrome P420_cam

Abstract

Cytochrome P450cam was subjected to high pressures of 2.2 kbar, converting the enzyme to its inactive form P420cam. The resultant protein was characterized by electron paramagnetic resonance, magnetic circular dichroism, circular dichroism, and electronic absorption spectroscopy. A range of exogenous ligands has been employed to probe the coordination structure of P420cam. The results suggest that conversion to P420cam involves a conformational change which restricts the substrate binding site and/or alters the ligand access channel. The reduction potential of P420cam is essentially the same in the presence or absence of camphor (-211 +/- 10 and -210 +/- 15 mV, respectively). Thus, the well-documented thermodynamic regulation of enzymatic activity for P450cam in which the reduction potential is coupled to camphor binding is not found with P420cam. Further, cyanide binds more tightly to P420cam (Kd = 1.1 +/- 0.1 mM) than to P450cam (Kd = 4.6 +/- 0.2 mM), reflecting a weakened iron-sulfur ligation. Spectral evidence reported herein for P420cam as well as results from a parallel investigation of the spectroscopically related inactive form of chloroperoxidase lead to the conclusion that a sulfur-derived proximal ligand is coordinated to the heme of ferric cytochrome P420cam.

A constitutively activated mutant of human soluble guanylyl cyclase (sGC): Implication for the mechanism of sGC activation

“…It has been suggested that carbonyl heme in cytochrome P420 and His-93-Cys myoglobin is pentacoordinated because of the loss of the weak thiolate coordination (40,41). Similar assumptions can be made about the ␣␤ Cys-105 sGC.…”

“…However, the position of the Soret peak after CO treatment is the same as for the pressure-induced cytochrome P420 (Table 2). Extensive EPR, magnetic circular dichroism, and Raman spectra analysis of cytochrome P420 demonstrated that, similar to cytochrome P450, the ferric heme is coordinated by the Cys-derived thiolate anion, although with a weakened ironsulfur ligation (increased FeOS stretch) possibly due to protonation of the thiolate anion (40). This comparison suggests that reconstituted ␣␤ Cys-105 sGC also has a pentacoordinated heme with Cys as the ligand, although with coordination weaker than in cytochrome P450.…”

The thermodynamics and kinetics of electron transfer in the cytochrome P450cam enzyme system

“…The formal potentia1 (at pH 7.0) of camphor-bound P450 cam has been reported as being approximately 3170 mV whereas that of the substrate-free enzyme has been reported as ranging from 3270 to 3330 mV [8,9]. The formal potential of putidaredoxin is 3235 mV [10] and therefore the reduction of substrate-free P450 cam should be thermodynamically unfavourable.…”

Novel Conformational Transitions of Human Cytochrome P450 2C8 during Thermal and Acid‐induced Unfolding

“…[10][11][12][13] In the denaturing process, the native cysteine coordination environment around the heme is changed, which is characterized by the conversion of P450 to its P420 form. The P420 form is termed according to the fact that its ferrous-CO absorbance maximizes at ca.…”

“…Most of previous unfolding/refolding studies were carried out on P450 cam , a soluble bacterial P450 comprising 414 amino acid residues. 10,11,[14][15][16][17][25][26][27] Some studies also used mammalian P450s (microsomal P450), 13,[28][29][30][31][32] however, the molecular details of these transitions had not been discussed in detail. In this paper, we studied human P450 2C8 to explore the P450 to P420 transition and its reversibility.…”