© 2003 Nature Publishing Group http://www.nature.com/naturestructuralbiology
ARTICLES
Ultrafast enzymatic reaction
dynamics in protochlorophyllide
oxidoreductase
a
Derren J Heyes1, C Neil Hunter1, Ivo H M van Stokkum2,
Rienk van Grondelle2 & Marie Louise Groot2
The reaction catalyzed by the light-driven enzyme protochlorophyllide oxidoreductase (POR) has been initiated with a
50-fs laser pulse. We show that the catalytic mechanism,
involving proton and hydride transfers, proceeds with time
constants of 3 ps and 400 ps. It is known that catalysis by POR
involves thermally excited protein dynamics; our results show
that these molecular motions occur on an ultrafast timescale.
Proton and hydride transfers are crucial to nearly all biological reactions. Their mechanisms cannot be understood until their intrinsic
timescales have been characterized; however, observation of these
events is often limited by diffusion rates of the reactants. Studies with
the light-driven chlorophyll biosynthetic enzyme NADPH:protochlorophyllide (Pchlide) oxidoreductase (POR; EC 1.3.1.33) can overcome this problem because the enzyme–substrate complex can be
formed in the dark and the reaction, which involves proton and
hydride transfers, can be observed in real time after catalysis is initiated with a 50-fs laser pulse. Therefore, POR may serve as an important model for enzymatic reaction dynamics. The enzyme catalyzes the
light-dependent trans addition of hydrogen across the C17–C18 double bond of Pchlide to produce chlorophyllide (Chlide)1, an important
regulatory step in the chlorophyll biosynthetic pathway and subsequent assembly of the photosynthetic apparatus2.
Here we report the first ultrafast data on POR and illustrate that
crucial steps in biological catalysis occur on the picosecond timescale.
Femtosecond pump-probe spectroscopy was used to follow the reaction catalyzed by POR from Synechocystis sp. PCC6803 via shifts in the
stimulated emission of the reactants. Measurements were performed
on a ternary Pchlide–POR–NADPH complex, on Pchlide alone and on
a site-directed mutant of POR in which Tyr189, the putative proton
donor3, had been replaced with phenylalanine.
The five time-gated absorbance difference spectra of the
Pchlide–POR–NADPH complex after a 50-fs laser pulse (Fig. 1a) show
that the POR enzyme displays reaction dynamics on an ultrafast
timescale. An initial increase of signal on the blue side (feature 1) of
the main band, originally centered at 642 nm, is followed by a progressive loss of amplitude of the main bleaching, particularly on the
red side (feature 2) of the band. These time-dependent spectral alterations are associated with the formation of a product in its excited
state, represented by a negative signal resulting from stimulated
b
Figure 1 Ultrafast reaction dynamics in POR. (a) Five representative spectra
spanning the nanosecond timescale of the experiment, sampled at various
times after a 50-fs laser pulse at 475 nm. Negative signals consist of
stimulated emission and bleaching of the ground state Pchlide absorption;
positive signals result from induced absorption from the excited state to
higher electronic states. The main spectral features are numbered. The
inset shows the ground state absorbance spectra of Pchlide and Chlide.
(b) Averaged absorption difference spectra at a 2.4-ns delay after excitation
for Pchlide–POR–NADPH, Pchlide–Y189F–NADPH and Pchlide alone. For
comparison, the spectra are normalized. All samples were produced as
described11,12 and were used at the following concentrations: POR, 420 µM;
Pchlide, 170 µM; NADPH, 1 mM; Y189F, 333 µM. The laser set-up has been
described13. Experiments were repeated four times on 4-ml samples that
flowed through a 1-mm cuvette; detection was at the magic angle (54.7°).
emission to the ground state at 674–677 nm. A comparison of the
absorption difference spectra of Pchlide–POR–NADPH, Pchlide alone
and Pchlide–Y189F–NADPH measured at the longest time delay of
2.4 ns (Fig. 1b) shows that bleaching of the Pchlide band in the
633–637 nm region occurred for all three complexes but product
formation was only observed for wild-type (wt) POR.
A more detailed analysis of the time-dependent data is presented in
the form of species-associated difference spectra (SADS) (Fig. 2a). The
three major features are an increase in signal on the blue side (feature 1)
of the 642 nm band, subsequent loss of amplitude on the red side
(feature 2) of this band and appearance of product. There is a 3-ps
phase of product formation, characterized by the appearance of a band
at 677 nm and an increase of signal on the blue side of the main 642 nm
band. The splitting of the dynamics, in which one part of the stimulated
emission blue shifts (feature d1) and another part red shifts to 677 nm,
1Department of Molecular Biology and Biotechnology, University of Sheffield, Sheffield S10 2TN, UK. 2Department of Exact Sciences, Vrije Universiteit, 1081 HV
Amsterdam, The Netherlands. Correspondence should be addressed to C.N.H. (c.n.hunter@shef.ac.uk).
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a
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b
indicates that part of the initial excited state evolves to a double-welled
potential energy surface. The blue shift of the stimulated emission indicates that the ground state potential energy surface of one of the wells
must be lower than the initial ground state. The other well is characterized by a smaller energy difference between ground and excited states.
One major feature of the next spectral evolution in 400 ps is a further
increase of the product state, slightly blue-shifting it, giving rise to the
band at 674 nm. This is accompanied by a marked loss of signal of the
main band (the negative feature of the diff 2 spectrum centered at
642 nm; feature d2). The equal but opposite amplitudes of the diff 1 and
diff 2 (see legend of Fig. 2 for definition) spectra between ∼625 nm and
∼636 nm (Fig. 2a) suggest that previously blue-shifted states (such as diff
1) can also evolve to the product state (diff 2) on this slower timescale.
Two further samples were studied and analyzed using three kinetic
components to provide assignments for the spectroscopic signals:
Pchlide alone and the Y189F sample, in which proton donation is not
possible (Y189F–NADPH–Pchlide). Although there was no productstate formation (Fig. 1b), some features of the spectral evolution seen
for wt POR were also observed for the Y189F mutant (Fig. 2b) but not
the Pchlide-only sample (data not shown). Specifically, the 660-ps
component shows decay in the red part of the main band at 640 nm
(see diff 2), as was observed for the native enzyme. The retention of
this component may indicate that hydride transfer from NADPH to
Pchlide can still occur in the mutant. Accordingly, the observed loss of
stimulated emission at 642 nm is likely to arise from hydride transfer
occurring on a timescale of 400–660 ps.
The remarkable appearance of an increased signal at shorter wavelengths within 3 ps, a specific feature of Pchlide bound to wt POR, is
not common in chromophore excited-state dynamics. The blueenhanced stimulated emission implies that the light-activated bound
Pchlide species is characterized by a lower ground state energy. The
excited state of this species may facilitate hydride transfer from the proS
face of NADPH to the C17 position3,4. In the Y189F mutant, which
Figure 3 Sequential and concerted mechanisms of Pchlide photoreduction.
2
Figure 2 SADS from a global analysis of the data using a sequential model.
In each case, the black spectrum evolves into the red spectrum, which then
evolves into the blue spectrum. The lifetimes of the blue spectra cannot be
accurately determined because our scan extends only to 2.4 ns. Doubledifference spectra (dotted) between the black/red (diff 1) and red/blue
components (diff 2) are shown to emphasize the spectral evolution.
(a) SADS of the ternary Pchlide–POR–NADPH complex. The black spectrum
evolves to the red spectrum in 3 ps, which evolves into the blue spectrum in
400 ps. The lifetime of the blue spectrum is 9 ns. (b) SADS of the
Pchlide–Y189F–NADPH mutant complex.
cannot donate a proton to Pchlide, the 3-ps gain on the blue side of the
642-nm band is negligible (Fig. 2a,b). The blue shift and gain of stimulated emission observed for wt POR, as well as their absence from the
proton transfer mutant, permit a provisional assignment of these signals to proton transfer. The 3-ps time constant for this proton transfer
is consistent with reports that the upper limit for intermolecular proton
transfer rate is 2.5 ps when it is not determined by diffusion5,6.
Formation of the product with two different reaction rates indicates
that two different routes for Pchlide photoreduction coexist: a concerted mechanism, where two bonds are broken simultaneously in the
transition state and the proton and hydride are transferred in a single
3-ps step, and a sequential mechanism, involving a 3-ps proton
transfer (giving rise to the blue-shifted species) followed by a ‘slow’,
400-ps hydride transfer (Fig. 3) that produces Chlide. Both sequential
and concerted proton-hydride transfer mechanisms have been suggested for enzyme-catalyzed reduction reactions based on theoretical
studies7,8. However, this is the first experimental report of hydride and
proton transfer in an enzyme occurring on a picosecond timescale.
Low-temperature spectroscopic measurements have revealed that the
POR catalytic mechanism consists of at least two steps: a lightdriven one occurring below the ‘glass transition’ temperature of proteins9, followed by one or more dark steps in which catalysis is assisted
by thermally excited protein dynamics10. Here we have shown that the
catalytic mechanism of POR, involving coupled proton and hydride
transfers, is complete within 400 ps. Taken together, these data show that
enzymatic reactions can involve molecular motions on a picosecond
timescale, which will aid an understanding of the mechanisms and time
constants of hydride and proton transfers in biological systems.
ACKNOWLEDGMENTS
D.J.H. and C.N.H. were supported by the BBSRC, UK. M.L.G. was supported by a
NWO-ALW PULS fellowship.
COMPETING INTERESTS STATEMENT
The authors declare that they have no competing financial interests.
Received 13 March; accepted 10 April 2003
Published online 5 May 2003; doi:10.1038/nsb929
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