Costas Varotsis

Gerald T. Babcock

Geurt Deinum

Johannes P. M. Schelvis

English

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Laser Chem., 1999, Vol. 19, pp. 223-225
Reprints available directly from the publisher
Photocopying permitted by license only
(C) 1999 OPA (Overseas Publishers Association) N.V.
Published by license under
the Harwood Academic Publishers imprint,
part of The Gordon and Breach Publishing Group.
Printed in India.
CO PHOTOLYSIS OF CYTOCHROME
OXIDASE INVESTIGATED BY PS
RESONANCE RAMAN SPECTROSCOPY
JOHANNES P. M. SCHELVIS a COSTAS VAROTSIS b
GEURT DEINUM a and GERALD T. BABCOCKa’ *
Department of Chemistry, Michigan State University,
East Lansing, MI 48824;
b Department of Chemistry, University of Crete, 71409, Iraklion, Crete, Greece
(Received 11 April 1997)
Low-power picosecond resonance Raman spectroscopy was used to investigate the
identity of the axial ligand of heme a3 and relaxation processes in the heme a3 pocket of.
cytochrome oxidase after CO photolysis. Our results show that the proximal histidine
remains ligated to heme a3 after CO photolysis excluding the transient ligation of a
photolabile, endogenous ligand. Furthermore, the relaxation of the heme a3 macrocycle
modes occurs on the sub ps time scale, while relaxation of the heme pocket to its
equilibrium conformation takes place on the laS time scale.
Keywords: Picosecond; resonance Raman; cytochrome oxidase; CO; photolysis
INTRODUCTION
Cytochrome oxidase contains 4 redox active metal centers: heme a,
heme a3, CUA and CuB. The catalysis takes place at the binuclear
center formed by heme a3 and CuB. The dynamics in the heme a3
pocket have been studied extensively using CO photolysis and two
models have been proposed. In.one model [1] the proximal histidine
remains ligated to heme a3, while in the other model [2] an endogenous
ligand dissociates from CuB and transiently binds to heme a3
*Corresponding author.
223
224 J. P. M. SCHELVIS et al.
displacing the proximal histidine after CO photolysis. We have applied
low-power ps resonance Raman difference spectroscopy to identify the
axial ligand of heme a3 directly after CO photolysis.
METHODS
Cytochrome oxidase was isolated from beef heart. Raman difference
spectroscopy was done with two synchronously pumped dye lasers
with 7 ps pulses at a 153 kHz repetition rate. Photolysis pulses were
36 nJ at 592 nm and probe pulses were 16 nJ at 441 nm.
RESULTS AND DISCUSSION
The low-frequency resonance Raman spectrum of the fully reduced
enzyme (Fig. a) contains contributions from both hemes, while the
spectrum of its CO-complex (Fig. lb) only shows peaks of heme a 2 +
using 441 nm excitation. The a+ vibrations that disappear upon CO
ligation, reappear in the Raman difference spectra (Fig. c-f) after
CO photolysis. Although the a+ macrocycle modes at 332, 366 and
400cm
-
are unchanged in the photolysis product, the Fe-his
frequency is upshifted from 214 to 220cm-1 as was observed in ns
experiments [1, 2]. The intensities of the modes in the difference spectra
were not sensitive to attenuation of the probe power by a factor of 8,
indicating that we do not observe additional photolysis due to the
probe pulses. Since the Fe-his mode is observed at 0 ps delay time and
substantial protein movement to accommodate ligation of a distal
ligand is not expected on this fast time scale, we conclude that the
2+proximal histidine remains ligated to heme a3. Furthermore, the a
macrocycle modes show no frequency shift on the time scale of our
experiment, so most likely they assume their equilibrium frequencies
on the sub ps time scale. The heme a3 pocket relaxes to its equilibrium
conformation on the gs time scale as indicated by the relaxation of the
Fe-his vibration.
CYTOCHROME OXIDASE 225
220 332 366 400
Ih ’,
a)
./
b) ’"
200 300 400
Raman Shift (cm-’)
FIGURE Low-frequency Raman spectra of cytochrome c oxidase from beef heart
obtained with 441 nm excitation. The equilibrium fully reduced enzyme (a) and its CO
complex (b). Difference spectra taken at 20 ps (c), 0 ps (d), 30 ps (e), and ns (f) after
CO photolysis. The difference spectra were obtained by subtracting the probe-only
spectrum from the pump-probe spectrum after normalization on the 438 cm- theme a
mode. In spectra lc-f, CO was photolyzed with 592 nm pulses.
References
[1] Findsen, E. W., Centen0, Babcock and Ondrias (1987). J. Am. Chem. Soc., 109,
5367.
[2] Woodruff, W. H., Einarsd6ttir, 0. et al. (1991). Proc. Natl. Acad. Sci. USA, 88,
2588.


CO Photolysis of Cytochrome
Oxidase Investigated by Ps
Resonance Raman Spectroscopy

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CO Photolysis of Cytochrome Oxidase Investigated by Ps Resonance Raman Spectroscopy

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