U
ltrafast
laser spectroscopy techniques are used to measure the
low-frequency vibrationa
l coherence spectra and nitric oxide rebinding kinetics of
Caldariomyces fumago ch
loroperoxidase (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 a
ll excitation wave
lengths, with a phase that is consistent with a ground-state Raman-excited vibrationa
l wavepacket. On the basis of a norma
l coordinate structura
l decomposition (NSD) ana
lysis, we assign this feature to the thio
late-bound heme doming mode. Spectra
l reso
lution of the probe pu
lse (“detuned” detection) revea
ls a mode at 349 cm
−1, which has been previous
ly assigned using Raman spectroscopy to the Fe−S stretching mode of native CPO. The ferrous species disp
lays a
larger degree of spectra
l inhomogeneity than the ferric species, as ref
lected by mu
ltip
le shou
lders in the optica
l absorption spectra. The inhomogeneities are revea
led by changes in the coherence spectra at different excitation wave
lengths. The appearance of a mode c
lose to 220 cm
−1 in the coherence spectrum of reduced CPO excited at 440 nm suggests that a subpopu
lation of five coordinated histidine-
ligated hemes is present in the ferrous state at a physio
logica
lly re
levant pH. A significant increase in the amp
litude of the coherence signa
l is observed for the resonance with the 440 nm subpopu
lation. Kinetics measurements revea
l that nitric oxide binding to ferric and ferrous CPO can be described as a sing
le-exponentia
l process, with rebinding time constants of 29.4
![](http://pubs.acs.org/images/entities/p<font color=)
lusmn.gif"> 1 and 9.3
![](http://pubs.acs.org/images/entities/p<font color=)
lusmn.gif"> 1 ps, respective
ly. This is very simi
lar to resu
lts previous
ly reported for nitric oxide binding to horseradish peroxidase.