Conformational Change Induced by Putidaredoxin Binding to Ferrous CO-ligated Cytochrome P450cam Characterized by 2D IR Spectroscopy

Abstract

The importance of conformational dynamics to protein function is now well-appreciated. An outstanding question is whether they are involved in the effector role played by putidaredoxin (Pdx) in its reduction of the O₂ complex of cytochrome P450cam (P450cam), an archetypical member of the cytochrome P450 superfamily. Recent studies have reported that binding of Pdx induces a conformational change from a closed to an open state of ferric P450cam, but a similar conformational change does not appear to occur for the ferrous, CO-ligated enzyme. To better understand the effector role of Pdx when binding the ferrous, CO-ligated P450cam, we applied 2D IR spectroscopy to compare the conformations and dynamics of the wild-type (wt) enzyme in the absence and presence of Pdx, as well as of L358P P450cam (L358P), which has served as a putative model for the Pdx complex. The CO vibrations of the Pdx complex and L358P report population of two conformational states in which the CO experiences distinct environments. The dynamics among the CO frequencies indicate that the energy landscape of substates within one conformation are reflective of the closed state of P450cam, and for the other conformation, differ from the free wt enzyme, but are equivalent between the Pdx complex and L358P. The two states co-populated by the Pdx complex are postulated to reflect a loosely bound encounter complex and a more tightly bound state, as is commonly observed for the dynamic complexes of redox partners. Significantly, this study shows that the binding of Pdx to ferrous, CO-ligated P450cam does perturb the conformational ensemble in a way that might underlie the effector role of Pdx.

Keywords: 2D IR spectroscopy; cytochrome P450; energy landscape; infrared spectroscopy; protein dynamics; putidaredoxin.

Figure 1

Structural model (PDB ID: 4JWU) of the complex of P450cam (blue) and Pdx (pink). The CO IR probe is circled in blue, the heme is shown in green, and the Fe atoms are shown as yellow spheres. Image generated with UCSF Chimera (Pettersen et al., 2004).

Figure 2

Linear FT IR spectra of heme-bound CO for (A) free wt, (B) L358P, and (C) the Pdx complex. Gaussian fits to the spectra are shown as shaded bands.

Figure 3

T_w-dependent 2D IR spectra of heme-bound CO for free wt (top row), L358P (middle row), and the Pdx complex (bottom row).

Figure 4

(A) CLS decays (points) with fits to exponential decays (lines) from analysis of 2D IR spectra assuming single component for free wt (blue), L358P (black), and Pdx complex (red). (B) CLS decays (points) with fits to exponential decays (lines) from analysis of 2D IR spectra in which the CLS decay at higher frequency was assumed equivalent to free wt (black) and that for the component at lower frequency was extracted for L358P (green) and Pdx complex (purple).

Figure 5

Overlay of (A) 2D diagonal slice (T_w of 0.25 ps) (blue) and FT IR spectrum (black) of Pdx complex and (B) 2D diagonal slices at T_w of 0.25 ps (blue), 20 ps (purple), 32 ps (red), and 44 ps (green).

Figure 6

Model illustrating the differences among the energy landscapes of the free wt, L358P, and Pdx complex based on the IR data. The conformational wells shaded in blue and red reflect the states associated with the bands at high and low frequency, respectively. The relative depths of the wells qualitatively reflect the differences in the population of the conformations. The breadth of the well reflects the inhomogeneous broadening in the state, and the height of the barriers among the substates are estimated from the different timescale of the dynamics.

Figure 7

Structural comparison of CO-ligated wt P450cam (green) and L358P (purple) showing CO, heme, camphor substrate, and several residues of the proximal and distal pockets. Protein crystal structures (PDB ID: 1T87 and 1T85) were superimposed using UCSF Chimera (Pettersen et al., 2004).