Low-frequency dynamics of Caldariomyces fumago chloroperoxidase probed by femtosecond coherence spectroscopy

Abstract

Ultrafast laser spectroscopy techniques are used to measure the low-frequency vibrational coherence spectra and nitric oxide rebinding kinetics of Caldariomyces fumago chloroperoxidase (CPO). Comparisons of the CPO coherence spectra with those of other heme species are made to gauge the protein-specific nature of the low-frequency spectra. The coherence spectrum of native CPO is dominated by a mode that appears near 32-33 cm(-1) at all excitation wavelengths, with a phase that is consistent with a ground-state Raman-excited vibrational wavepacket. On the basis of a normal coordinate structural decomposition (NSD) analysis, we assign this feature to the thiolate-bound heme doming mode. Spectral resolution of the probe pulse ("detuned" detection) reveals a mode at 349 cm(-1), which has been previously assigned using Raman spectroscopy to the Fe-S stretching mode of native CPO. The ferrous species displays a larger degree of spectral inhomogeneity than the ferric species, as reflected by multiple shoulders in the optical absorption spectra. The inhomogeneities are revealed by changes in the coherence spectra at different excitation wavelengths. The appearance of a mode close to 220 cm(-1) in the coherence spectrum of reduced CPO excited at 440 nm suggests that a subpopulation of five coordinated histidine-ligated hemes is present in the ferrous state at a physiologically relevant pH. A significant increase in the amplitude of the coherence signal is observed for the resonance with the 440 nm subpopulation. Kinetics measurements reveal that nitric oxide binding to ferric and ferrous CPO can be described as a single-exponential process, with rebinding time constants of 29.4 +/- 1 and 9.3 +/- 1 ps, respectively. This is very similar to results previously reported for nitric oxide binding to horseradish peroxidase.

Figure 1

Spatial view of the active site of chloroperoxidase, including the Mn²⁺ ion and its binding site (10) (Protein Data Bank entry 1CPO). Cys29 is the proximal ligand. The distal pocket features Phe186 and Glu183 as residues that affect the catalytic cycle. Mn²⁺ is coordinated by one of the heme propionates, three amino acids (Glu104, His105, and Ser108), and two water molecules (not shown). Two histidine residues (His105 and His107) that are positioned close to the distal pocket are also shown.

Figure 2

Electronic absorption spectra of native ferric CPO (red), reduced CPO (black), ferric P450_CAM (blue), and reduced P450_CAM (green). The CPO spectra were recorded at pH 5 in 0.1 M phosphate buffer, and the maximum of the Soret peak is 399 nm for the ferric species and 409 nm for the reduced species. The camphor-bound P450_CAM spectra were measured at pH 7 in 0.1 M phosphate buffer, and the maximum of the Soret peak is 392 nm for the ferric species and 414 nm for the reduced species. The spectra have been normalized using published extinction coefficients.

Figure 3

Correlations between the resonance Raman and coherence spectra of native (ferric) CPO. The top trace is the Raman spectrum measured with an excitation wavelength of 413.1 nm. The middle trace is the detuned coherence spectrum (the inset shows the coherent oscillations and LPSVD fit). The bottom trace is the open band coherence spectrum, and the inset again shows the coherent oscillations and LPSVD fit under this condition. The coherence spectra were measured using excitation at 425 nm. The dispersed data were measured with a 0.5 nm spectral window, detuned 5 nm to the blue of the carrier frequency maximum (i.e., λ_pr = 420 nm).

FIGURE 4

Open band coherence measurements of native ferric CPO at pH 5. The left panels show the oscillatory signal (○) and their LPSVD fits (—). Right panels show the corresponding LPSVD-generated power spectra. Experiments were carried out with ~60 fs pulses at 410, 415, 420, and 425 nm.

Figure 5

Comparison of low-frequency coherence spectra of three different cysteine-ligated ferric heme proteins [CPO, P450_CAM (CYP101) with camphor bound, and thermophyllic P450 (CYP119)] and two histidine-ligated ferric heme proteins (HRP and metmyoglobin). The left panels present the oscillatory signal and the LPSVD fit. The right panels show the corresponding LPSVD power spectra. The open band experiments were carried out at wavelengths specified in the right panels using ~60 fs pulses.

Figure 6

Correlations between the resonance Raman and coherence spectra of ferrous CPO. The top trace is the Raman spectrum measured with a 413.1 nm excitation wavelength. The middle trace is the detuned coherence spectrum (the inset shows the coherent oscillations and LPSVD fit). The bottom trace is the open band coherence spectrum, and the inset again shows the coherent oscillations and LPSVD fit under this condition. The coherence spectra were measured using 420 nm excitation. The dispersed data were measured with a 0.5 nm spectral window, detuned 5 nm to the blue of the carrier frequency maximum (i.e., λ_pr = 415 nm).

Figure 7

Open band measurements of reduced CPO at pH 5. The left panels show the oscillatory signal (○) and their LPSVD fits (—). The right panels present the corresponding LPSVD-generated power spectra. Experiments were carried out with ~60 fs pulses at 420, 425, and 440 nm. The increase in the relative amplitude of the oscillatory signal as well as changes in the pattern of frequencies should be noted as the excitation wavelength is tuned to 440 nm.

Figure 8

Transient pump probe signal of reduced CPO at 440 nm. Note the strong vibrational coherence signal. The full transmission change is shown in the top panel on a logarithmic scale. The quantity ΔT_max is defined by the maximal probe transmission change, which usually occurs near the 60-80 fs time delay. The bottom panel shows the oscillatory component of the signal (top curve) obtained after removal of the exponentially decaying component from the raw data. The LPSVD-extracted oscillations of the two strongest modes (88 and 130 cm^-1) are also displaced below for easier visualization.

Figure 9

Correlations between the resonance Raman and coherence spectra of ferrous CPO. The top trace is the Raman spectrum measured with an excitation wavelength of 442.1 nm. The middle trace is the detuned coherence spectrum (the inset shows the coherent oscillations and LPSVD fit). The bottom trace is the open band coherence spectrum, and the inset again shows the coherent oscillations and LPSVD fit under this condition. The coherence spectra were measured using 440 nm excitation. The dispersed data were measured with a 0.5 nm spectral window, detuned 5 nm to the blue of the carrier frequency maximum (i.e., λ_pr = 435 nm).

Figure 10

Comparison of the low-frequency coherence spectra of three different cysteine-ligated ferrous heme proteins [CPO, P450_CAM (CYP101) with camphor bound, and thermophyllic P450 (CYP119)] and two histidine-ligated ferrous heme proteins (reduced HRP and deoxymyoglobin). The left panels present the oscillatory signal and the LPSVD fit. The right panels show the corresponding coherence spectra. Experiments were carried out at wavelengths specified in the right panels using ~60 fs pulses.

Figure 11

NO rebinding kinetics for the ferric and ferrous forms of CPO. The data are fit with a single-exponential function with a constant background. The fitting parameters and fundamental geminate rebinding (k_BA) and escape (k_out) rates are listed in Table 2.

Figure 12

NSD analysis in the mass-weighted coordinate space for (A) ferric and (B) ferrous heme proteins (CPO, CYP101, CYP119, HRP, and Mb). The displacement in amu^1/2 Å is the square root of the sum of squares of the mass-weighted displacements from planarity for Fe and the 24 porphyrin (4 N and 20 C) atoms. The key out-of-plane modes are denoted as follows: pro, propellering (blue); ruf, ruffling (green); sad, saddling (red); wav(x), waving_x (light blue); wav(y), waving_y (yellow); dom, doming (purple); and invdom, inverse doming (gray). A negative displacement is defined only for doming and inverse doming to indicate the direction of the Fe displacement (+, proximal; -, distal). The PDB entries used to determine the structures are 1CPO, 1DZ4, 1IO9, 1ATJ, 1BZ6, 2J19, 1DZ6, 1H58, and 1BZP for CPO(Fe³⁺), CYP101(Fe³⁺), CYP119(Fe³⁺), HRP(Fe³⁺), Mb(Fe³⁺), CPO(Fe²⁺), CYP101(Fe²⁺), HRP(Fe²⁺), and Mb(Fe²⁺), respectively. The crystal structure for CYP119(Fe²⁺) is not available.