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INTRODUCTION |
Weak far-red chemiluminescence is emitted when
Mn2+-activated
rubisco1 catalyzes its
oxygenase reaction (1). Neither the physiological cofactor,
Mg2+, nor any other metal ion has been found to support
observable luminescence. An incomplete spectrum, based on the (then)
unproven assumption that the spectrum did not change as the oxygenase
reaction proceeded, exhibited characteristics typical of
Mn2+ luminescence (2). Cox et al. (3)
investigated the effects of potential inhibitors and enhancers and
found no evidence to support the suggestion (1) that singlet
O2 has a role in this luminescence. However, the
Mn2+-rubisco luminescence is weak, with quantum yield
estimated at 10
7 to 10
9 photons per
turnover (2). This leaves open the possibility that some infrequent
side reaction of the oxygenase reaction of Mn2+-activated
rubisco, undetectable by other means, might generate singlet
O2, which contributes to the chemiluminescence directly or
indirectly by a sensitization mechanism (4).
Here we perform a direct spectroscopic test for the production of
singlet O2 by this system, seeking to observe the emission at 1268 nm associated with its monomolecular transition to the triplet
O2 ground state. This emission is much more intense than that arising from the dimolecular process resulting from encounters between two molecules of singlet O2. The latter generates
emissions at 633 and 703 nm that are detected more readily by
photomultipliers. Furthermore, the 1268 nm luminescence is independent
of the concentration and lifetime factors that determine the emission
from such encounters. Therefore, the presence of 1268 nm emission can
reveal low levels of singlet O2 in biological systems (5).
Previously, enzyme-associated emission of far-red light at wavelengths
longer than 1100 nm confirmed the production of singlet O2
by lactoperoxidase (6), chloroperoxidase and catalase (7). On the other
hand, chemiluminescence by acetolactate synthase detectable with a
photomultiplier was attributed to the dimolecular reaction (8), but the
absence of detectable IR emission did not support the involvement of
singlet O2 (9).
Prompted by observations that the luminescence yield appeared to differ
between rubiscos from different sources (3), we here determine a more
complete and less equivocal spectrum of the chemiluminescence. The
spectrum reported previously for spinach rubisco (2) was subject to
spectrophotometric constraints, including the need to correct for decay
of enzyme activity during scanning. Here, we use a very sensitive
spectrograph to accumulate the entire spectrum in a few seconds. This
enables the measurement of spectral differences in the emission between
rubiscos from different sources.
 |
EXPERIMENTAL PROCEDURES |
Enzyme and Substrate Preparation--
Rubisco was purified from
spinach (Spinacea oleracea L.) leaves by anion exchange
chromatography (10). Rubisco from wild type tobacco (Nicotiana
tabaccum L.) and its L335V mutant (11) were purified by
crystallization (12). The rbcM gene from
Rhodospirillum rubrum, encoding an N-terminal hexahistidine
tag, was expressed in Escherichia coli and the recombinant
protein was purified by nickel-nitrilotriacetic acid-agarose
chromatography using the manufacturer's procedure (Qiagen). Purified
rubisco was stored at
80 °C in 50 mM NaCl, 10 mM sodium phosphate, 1 mM EDTA, 10% (v/v)
glycerol, pH 7.6. The total concentration of rubisco active sites was
determined by
[14C]2'carboxy-D-arabitinol-1,5-bisphosphate
binding (13, 14). RuBP was synthesized by the procedure of Horecker
et al. (15) and purified by Dowex 1-Cl chromatography
(16).
Survey of Visible and IR Emission Wavelengths--
A
dual-detector apparatus with a Hamamatsu R943-02 photomultiplier and an
ADC 403L germanium detector was used to measure a wide range of visible
and IR wavelengths (Fig. 1). The relative responses of the two
detectors were established from the manufacturer's specifications and
spectral measurements in the range from 600 to 1000 nm. Data for the
spectral sensitivity of the germanium detector were extended beyond the
range provided by the manufacturer (1200-1800 nm) to the important
700-1200 nm region by measuring a black-body source dispersed through
a monochrometer. A correction for the variation in diffraction
efficiency of the grating with wavelength was taken from data provided
by the grating manufacturer (Richardson Grating Laboratories).
Reaction mixtures (1 ml) in a quartz cuvette were monitored
simultaneously by both detectors (Fig. 1). For experiments with rubisco
using H2O as solvent, the spinach enzyme (76 µM active sites) was preactivated by incubation for 30 min in 25 mM Tris-HCl or EPPS-NaOH, pH 8.1, 0.1 mM EDTA, 10 mM NaHCO3, 2 mM MnCl2 (Aldrich). The reaction mixture
contained 3.1 µM active sites of activated rubisco, 25 mM Tris (adjusted to pH 8.1 with HCl), 0.1 mM
EDTA, 0.4 mM NaHCO3, and 2 mM
MnCl2 and the reaction was started by addition of RuBP to
390 µM. For experiments with 2H2O
as solvent, spinach rubisco (3.1 µM active sites) was
preactivated for 10 min in 25 mM Tris-HCl, 0.19 mM EDTA, 1.9 mM MnCl2, 3.9 mM NaHCO3, 97.9% 2H2O.
The pD was 8.2, obtained by mixing calculated weights of Tris base and
Tris-HCl. The reaction was started by the addition of RuBP (to 190 µM).
Lactoperoxidase (Sigma, from bovine milk, lyophilized) was dissolved
(0.1 mg ml
1) in 20 mM acetic acid-NaOH, pH
4.5. The enzyme solution (1 ml) was added first to the cuvette,
followed by 0.1 ml of 200 mM NaBr. After thorough mixing,
0.1 ml of 200 mM H2O2 was pipetted
onto the top of the solution in the cuvette without additional mixing.
Determination of the Chemiluminescence Spectrum--
An f4 0.5m
spectrograph was constructed using a back-thinned, deep-depleted,
charge-coupled detector (CCD, supplied by Roper Scientific) and a
low-dispersion grating blazed at 800 nm. Each spectrum (650-890 nm)
was accumulated over 20 s. The spectra were calibrated and
corrected for the wavelength dependence of the sensitivity of the CCD
by reference to black-body radiation from a tungsten filament of known
temperature (determined by an optical pyrometer, Leeds and Northrup
8627). The light from the center of the tungsten filament was
attenuated uniformly by passing it through a low-duty cycle chopper
before being focused on the spectrometer slit. Details about reaction
mixtures and other procedures are given in the legends.
 |
RESULTS |
Survey of Visible and IR Emissions--
Two detectors were
required to cover the visible/IR range of wavelengths of interest to
this study. The sensitivity of the photomultiplier was greatest in the
visible region, declining in the IR. By contrast, the germanium
detector was most sensitive in the IR beyond 1200 nm, with declining
sensitivity toward the visible. Using the dual-detector system (Fig.
1) without filters, luminescence was
detected by both the photomultiplier and the germanium detector while
rubisco was catalyzing the oxygenation of RuBP (Table
I). A filter that blocked all radiation
with wavelengths shorter than 1000 nm reduced the signal from the
germanium detector by over 90%; a filter that blocked all wavelengths
shorter than 1200 nm eliminated it entirely. The nature of the buffer
used in the experiments had no effect on the intensity of the
luminescence; identical results were obtained with Tris-HCl and
EPPS-NaOH.

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Fig. 1.
Schematic representation of the dual-detector
apparatus. Total luminescence emitted during oxygenation was
monitored simultaneously with a red-sensitive, high-gain
photomultiplier (sensitive to wavelengths from 200 to 900 nm) and a
cooled germanium detector (most sensitive to wavelengths between 1100 and 1700 nm, but with low level sensitivity to wavelengths as short as
700 nm) positioned on opposite sides of the source. The source was a
1-cm2 quartz cuvette containing enzyme reaction mixtures.
Filters were interposed between the source and the detectors to select
the required wavelength ranges.
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Table I
Photomultiplier (PM) and germanium detector outputs for a range of
experimental conditions and filter sets
Spinach rubiosco was used.
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When the solvent was 98% 2H2O, the signal at
both detectors doubled and a filter that blocked wavelengths shorter
than 1100 nm eliminated the signal from the germanium detector.
Calibration with a black-body source showed that the germanium detector
was approximately one-tenth (±20%) as sensitive at 800 nm (the peak of the rubisco emission, see later) as at 1200 nm. Because the minimum
observable signal of the germanium detector was about 0.01 mV, more
than 99% of the signal measured for rubisco chemiluminescence in
2H2O occurred at wavelengths shorter than 1100 nm (Table I). Hence radiation emitted by the rubisco samples at
wavelengths longer than 1100 nm must be <0.1% of the total
chemiluminescence intensity.
As a positive control, a very strong, short-lived luminescence signal
was measured at wavelengths longer than 1200 nm by the germanium
detector when singlet O2 was generated by lactoperoxidase. However, at the same time, no radiation was detected by the
photomultiplier at wavelengths longer than 650 nm (Table I).
Spectrum of Rubisco Luminescence--
Although both detectors of
the dual-detector system had significant sensitivity over the 700-850
nm range of wavelengths, the extremely low intensity of rubisco
chemiluminescence and its decay with time precluded accurate
determination of the spectrum with these detectors. A spectrograph
based on a CCD detector was constructed for this purpose.
The output from the CCD spectrograph required correction for the
wavelength dependence of the sensitivity of the CCD detector. An
example of a spectrum of the luminescence from
Mn2+-activated spinach rubisco before and after correction
is shown in Fig. 2. The intensity of
chemiluminescence for each pixel of the detector, accumulated over
20 s, is expressed as the specific intensity per nanomole of
rubisco active sites present in the cuvette. The correction was
greatest at the longer wavelengths, as expected, and increased the
scatter of the spectral data (Fig. 2b). Corrected spectra
for spinach, tobacco, and R. rubrum rubiscos are
shown in Figs. 3 and 4. When the
substrate RuBP was present in excess, the
chemiluminescence exhibited a slow single-phase exponential decay (Fig.
4, a and b). When the reaction conditions were
such that the RuBP was fully consumed eventually (Figs. 3 and
4c), this exhaustion was accompanied by a further decay in the luminescence to zero.

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Fig. 2.
Emission spectrum of chemiluminescence by
spinach rubisco. The abscissa is linear in terms of
wave number. Spinach rubisco (77.5 µM active sites) was
activated for 30 min in 25 mM Tris-HCl, 0.1 mM
EDTA, 2 mM MnCl2, 10 mM
NaHCO3, pH 8.1. The activated rubisco was transferred to a
reaction mixture containing a solution saturated with O2 by
bubbling. The final concentrations were 3.1 µM rubisco,
24 mM Tris-HCl, 0.1 mM EDTA, 2 mM
MnCl2, 0.4 mM NaHCO3 and the
reaction was started by the addition of RuBP (to 390 µM).
Accumulation of the spectrum by the CCD apparatus (see "Experimental
Procedures") commenced 5 s later and continued for 20 s.
a, uncalibrated spectral data. b, data after
correction for variation in the sensitivity of the CCD with wavelength.
The fitted Gaussian regression curve had a mean (dotted
line) of 797 nm and r2 of 0.963.
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Fig. 3.
Emission spectra and time course of peak
intensity (inset) of chemiluminescence by spinach rubisco in
1H2O (a) and 96.5% (v/v)
2H2O (b). Spinach rubisco
(7.6 µM sites) was preincubated for 10 min in 24 mM Tris-HCl, 0.19 mM EDTA, 1.9 mM
MnCl2, 3.9 mM NaHCO3 in
1H2O or 2H2O. The pH/pD
was 8.2, obtained by mixing calculated weights of Tris base and
Tris-HCl. The reaction was started by the addition of RuBP (to 190 µM) and accumulation of the first spectrum commenced
5 s later. The spectra were accumulated over consecutive 20 s
intervals and those (from top down) for 5-25, 85-105,
125-145, and 165-185 s are shown. The Gaussian regression curves
fitted to the 5-25 s spectra had means (dotted lines) and
r2 of 800 nm and 0.991 (1H2O) and 799 nm and 0.996 (2H2O), respectively. The time courses
(insets) show the peak chemiluminescence intensity for all
spectra collected against the midpoint time during collection of each
spectrum and the line represents the regression for
single-phase exponential decay.
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Fig. 4.
Emission spectra and time course
(inset) of chemiluminescence by rubisco.
a, wild type tobacco rubisco in
1H2O. Enzyme (99 µM sites) was
activated for 10 min at 50 °C, cooled to 20 °C, and then
maintained at that temperature for about 30 min in 25 mM
Tris-HCl, 0.2 mM EDTA, 2 mM MnCl2,
10 mM NaHCO3, pH 8.1, before transfer to the
assay. The reaction mixture (1.0 ml) contained 7.9 µM
rubisco, 25 mM Tris-Cl, 0.1 mM EDTA, 2 mM MnCl2, 0.8 mM
NaHCO3, pH 8.1, and the reaction was started by the
addition of RuBP (to 770 µM). The spectra were
accumulated over consecutive 20 s and those (from top
down) for 5-25, 85-105, 185-205, and 585-605 s are shown. The
Gaussian regression curve fitted to the 2-25 s spectrum had a mean
(dotted line) of 798 nm and r2 of
0.987. The time course (inset) shows the peak
chemiluminescence intensity for all spectra collected against the
midpoint time for collection of each spectrum and the line
represents the regression for single-phase exponential decay.
b, emission spectra and time course of chemiluminescence by
the L335V mutant of tobacco in 1H2O. Enzyme
(110 µM) was activated as for a in 12 mM Tris-HCl, 0.1 mM EDTA, 2 mM
MnCl2, 10 mM NaHCO3, pH 8.1, before
transfer to the assay. The reaction mixture (1.0 ml) contained 8.5 µM rubisco, 22 mM Tris-Cl, 0.17 mM EDTA, 2 mM MnCl2, 0.8 mM NaHCO3, pH 8.1, and the oxygenase reaction
was started by the addition of RuBP (to 770 µM). The
spectra were accumulated over consecutive 20 s and those (from
top down) for 5-25, 65-85, and 85-305 s, are shown. The
Gaussian regression curve fitted to the 5-25 s spectrum had a mean
(dotted line) of 800 nm and r2 of
0.988. The time course (inset) shows the peak
chemiluminescence intensity for all spectra collected against the
midpoint time for collection of each spectrum. c, emission
spectra and time course of chemiluminescence by R. rubrum
rubisco in 1H2O. Enzyme (6.2 µM
in 500 µl) was dialyzed against 25 mM Tris-HCl, 0.1 mM EDTA, 2 mM dithiothreitol, pH 8.1. Dialyzed
enzyme was preincubated for 30 min in 23 mM Tris-HCl, 0.1 mM EDTA, 1.8 mM dithiothreitol, 2 mM MnCl2, 40 mM NaHCO3,
pH 8.1. The reaction mixture (1.0 ml) contained 2.7 µM
rubisco, 24 mM Tris-HCl, 0.1 mM EDTA, 1.1 mM dithiothreitol, 2 mM MnCl2, 22 mM NaHCO3, pH 8.1, and the oxygenase reaction
was started by the addition of RuBP (to 770 µM). The
spectra were accumulated over consecutive 20 s and those (from
top down) for 2-25 and 45-65 s are shown. The Gaussian
regression curve fitted to the 5-25-s spectrum had a mean
(dotted line) of 760 nm and r2 of
0.870. The time course (inset) shows the peak
chemiluminescence intensity for all spectra collected against the
midpoint time for collection of each spectrum.
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The luminescence spectrum is modeled closely, but not quite perfectly
(r2 values in figure legends), by a Gaussian
curve, symmetrical in terms of wave number. The fitted line is shown
for the first and most intense corrected spectrum accumulated between 5 and 25 s after addition of RuBP to the reaction mixtures (Figs.
2-4). With time, the luminescence diminished in intensity but the
spectrum was unchanged.
The wavelength of peak emission, estimated from the fitted curves for
spinach rubisco in H2O, was 797 nm (Fig. 2b) or
800 nm (Fig. 3a). A similar spectrum, peaking at 799 nm, was
observed for spinach rubisco in 2H2O (Fig.
3b), but the intensity approximately doubled. The
chemiluminescence decayed to zero when the RuBP had been consumed
completely. This took about 170 s for rubisco in H2O
and about 200 s in 2H2O (Fig. 3), showing
that the total rubisco activity (carboxylase plus oxygenase) was
slightly lower in 2H2O. Rubisco from wild type
tobacco (Fig. 4a) exhibited a chemiluminescence spectrum
indistinguishable in profile from that of spinach rubisco, with peak
emission at 798 nm. The peak wavelength of the emission from the L335V
mutant of tobacco rubisco was similar (800 nm), but the intensity was
approximately halved (Fig. 4b).
The spectrum of the chemiluminescence emitted by rubisco from R. rubrum (Fig. 4c) was blue-shifted compared with that of
the enzymes from higher plants, with peak emission at 760 nm. The specific intensity per nanomole of active sites was also smaller.
Photoluminescence Could Not Be Induced by Laser Excitation--
We
attempted to excite photoluminescence from Mn2+-activated
rubisco by laser excitation. Some weak, variable emission was indeed seen, as well as some Raman features. The emission could not be identified clearly with the chemiluminescence measured. Although previous attempts to measure Mn2+ emission using aqueous
inorganic solutions of Mn(II) have failed (17), we have observed a
number of examples of laser-excited photoluminescence from aqueous
Mn2+ with the same apparatus used in this
study.2 However, the process
is remarkably feeble, requiring very high (5 M)
managanese concentrations. The Mn2+ concentrations
in our rubisco samples were far lower (up to 2 mM
active-site concentrations, requiring purified rubisco at up to 140 mg
ml
1), precluding direct photoexcitation of measurable
photoluminescence. Laser excitation of other chromophoric units in the
protein does not lead to efficient electronic excitation transfer to
the manganese. Consequently, the luminescence spectrum is dominated by
other (weak) emitters.
 |
DISCUSSION |
The Spectral Data Eliminate Singlet O2 as a Possible
Source of Luminescence--
The germanium detector of the
two-detector, visible/IR measurement system (Fig. 1) detected intense
luminescence at wavelengths longer than 1200 nm when the
lactoperoxidase reaction, a known producer of singlet O2,
was used (Table I). This luminescence is attributable to the
monomolecular decay of singlet O2 (1268 nm) (7). On the
other hand, the photomultiplier, whose sensitivity extends from visible
wavelengths into the IR to ~900 nm, detected no emission from the
same system. Therefore, we conclude that luminescence caused by the
dimolecular dismutation of singlet O2 pairs (expected to
show peaks at 634 and 703 nm (4)) is negligible in this system, even
when the monomolecular reaction is readily detectable. The germanium
detector detected no luminescence from manganese-rubisco at wavelengths
longer than 1100 nm. Even in 98% 2H2O, which
greatly enhances chemiluminescence from enzyme-generated singlet
O2 (19) no emission from manganese-rubisco was detected at
wavelengths longer than 1100 nm (<0.1% of that detected by the
photomultiplier at shorter wavelengths). Therefore, the emission from
manganese-rubisco detected by the photomultiplier cannot originate from
the dimolecular singlet-O2 reaction. Consistent with this
conclusion, the spectra of the Mn2+-rubisco luminescence
recorded by the CCD detector showed no sign of any features at 634 and
703 nm, even in 98% 2H2O (Fig. 3).
The Luminescence Is Characteristic of Octahedral
Mn2+--
The broad smooth spectra of the luminescence
from Mn2+-activated rubisco resembles the emission from
six-coordinate Mn2+ compounds. Manganese luminescence
exhibits spectra with peak wavelengths depending strongly on the local
environment of the metal ion and the resultant crystal (ligand) fields
(20). Non-enzymic reactions involving an excited Mn2+
species show such broad emission spectra. In a set of inorganic reactions involving Mn3+ reduction to Mn2+, the
peak emission was at either 689 or 730 nm (17), whereas luminescence
from the oxidation of glyoxal by Mn(III)-lactate chelate peaked at
about 710 nm (21). These systems also lacked any emission attributable
to singlet O2.
An alternative conceivable source of the rubisco chemiluminescence that
must be considered is the possible involvement in the oxgenase reaction
mechanism of a dioxetane intermediate involving carbons 2 and 3 of the
RuBP substrate. Although such a mechanism was excluded for the main,
Mg2+-dependent oxygenase pathway by
18O2 labeling studies (22), the possibility
that a trace of the reaction flux passes through a dioxetane
intermediate cannot be excluded, particularly when Mn2+ is
substituted as the active-site metal. Dioxetanes are known to luminesce
during coupled O-O and C-C bond cleavage but usually at much shorter
wavelengths than observed with rubisco. Dioxetane emissions at
wavelengths longer than 540 nm are rare. Only in the case of chemically
initiated electron exchange luminescence of phenolate-substituted
dioxetanes has longer wavelength light been observed, and even then the
maximum wavelengths were shorter than 630 nm (23).
Higher plant rubisco-manganese emits at substantially longer
wavelengths than the laser-excited luminescence from hexa-aquo Mn(II),
which has a maximum at 770 nm.2 In a precisely octahedral
field with six identical ligands, the manganese emission corresponds to
a 4T1g
6A1g
transition (both spin and parity forbidden) within the weak field
d5 manifold of Mn(II). If the ligand field is
increased, the energy of this transition decreases. Thus, in
higher plant rubisco-manganese, either the ligating atoms at the
manganese site are slightly stronger ligands than the oxygen of
H2O or the local field has significantly lower symmetry. In
the latter case, an inequivalence of the ligands, either in nature or
deviation from precise octahedral position, leads to a splitting of the
4T1 state. Only the lowest excited state can
emit and then the emission seen from the distorted system could thus
appear as a lower energy component of the split
4T1 state. These possibilities could be
distinguished by measurement of the absorption energies and
characteristics of the rubisco-manganese site. Although the Mn(II)
transitions are not easily seen directly by absorption spectroscopy
because of their intrinsic weakness, they may be made visible in
temperature-dependent magnetic circular dichroism
spectroscopy, which is a particularly sensitive and selective method
for observing paramagnetic metalloenzyme centers. Previous ESR studies
have demonstrated varying degrees of rhombic distortion of the
coordination sphere of rubisco-bound Mn2+ in enzymes from
spinach and R. rubrum complexed with the six-carbon intermediate analog,
2'-carboxy-D-arabinitol-1,5-bisphosphate (24-26).
Furthermore, x-ray crystallography studies showed that the bound
Mg2+ ion in activated spinach rubisco is in close to
octahedral coordination when the enzyme is not ligated by sugar
phosphate, but that considerable distortion of the metal ion
coordination sphere occurs after binding of
2'-carboxy-D-arabinitol-1,5-bisphosphate (27).
The Manganese Luminescence Spectrum of Hexadecameric
Rubisco--
The intensity of successive spectra diminished
exponentially with time when the RuBP concentration was far in excess
of the Km (400 nM for the spinach enzyme
(2)). This well known "fallover" property of rubisco results from
the tight binding of inhibitors originating both from rubisco side
reactions and from
D-glycero-2,3-pentodiulose-1,5-bisphosphate, a contaminant of RuBP (16). No change in the spectral peak or distribution was
apparent with time. This eliminates one of the potential criticisms of
the spectrum reported by Lilley et al. (2). In that study, the spectra were recorded much more slowly than in the present study
and required correction for the time-dependent decay of the
luminescence; a correction that assumed that the spectrum did not
change with time. The unchanging spectra also suggest that the presence
of such tight-binding inhibitors on one or more of the active sites
does not affect the orientation of amino acid residues that coordinate
Mn2+ ions within the remaining unbound and catalytically
active sites on the same rubisco hexadecameric holoenzyme.
The spectra for rubisco from spinach, tobacco wild type, and tobacco
L335V mutant are indistinguishable in terms of general shape and maxima
in the 797-800 nm range. Although spinach and tobacco rubisco differ
by several amino acid residues in the large and small subunits, the
residues involved in their active sites are the same and they have
similar structures (28) and kinetic characteristics. L335V rubisco
(Mg2+-activated) has an oxygenase
Vmax 33% of the wild type and a
CO2/O2 specificity ratio 25% of the wild type
(11). The L335V mutation represents a shortening of the side chain at
this residue, which does not participate directly in catalysis, but may
contribute to positioning the mobile loop 6 as its terminal methyl
groups contact the P2 phosphate of bound RuBP in the active site. This has the potential to cause a small change in the position or
orientation of the
amino group of the adjacent Lys-334 that is
catalytically critical (11). This group is involved in hydrogen bonding
with an intermediate carboxyl group formed by the attack of
CO2 on the enediol form of RuBP. Here, the L335V rubisco,
when Mn2+-activated, exhibited 52% of the
chemiluminescence intensity of the wild type. However, the absence of
any perceptible effect on the spectrum suggests that the ligand field
of Mn2+ at the active site is not disturbed by the L335V mutation.
When the reaction was performed in 96.5% 2H2O,
the intensity was approximately doubled as reported previously (1, 3). However, the peak wavelength (799 nm) and distribution of the emission
was unchanged.
Difference in Luminescence between Dimeric and Hexadecameric
Rubiscos--
The spectrum for R. rubrum rubisco, a
homodimer devoid of small subunits, has a maximum at 760 nm, clearly
blue-shifted in comparison to that from the hexadecameric higher plant
enzymes composed of both large and small subunits. Indeed, this peak is located at a wavelength slightly shorter than photoluminescence from
hexa-aquo Mn(II).2 Although the aminoacyl side chains near
the metal ion are conserved between the rubisco types, the dispositions
of these critical ligand-forming groups may be slightly altered by
substitutions in residues a little more removed, or by the absence in
the dimeric enzyme of small subunits. The latter influence the
catalytic activity of higher plant rubiscos by remote interactions,
which telegraph structural alterations to the active site (29). ESR
spectral differences between the
Mn2+-2'-carboxy-D-arabinitol-1,5-bisphosphate
complexes of the plant and R. rubrum enzymes also indicate
differences in metal coordination between the two types of rubisco (24,
25).
Temperature and pH have a marked effect on the relative luminescence
yield of R. rubrum rubisco, unlike the situation with hexadecameric enzyme (3). The temperature response of the dimeric enzyme was attributed to temperature sensitivity of the balance between
radiationless and luminescent decay pathways and the pH effect to the
presence in the dimeric, but not the hexadecameric, enzyme of an
active-site group with pKa above 7.4. Clearly this
group influences the Mn2+ ligand field.
The comparatively lower intensity of luminescence from R. rubrum rubisco (Fig. 4c) is partly because of
competitive inhibition of the oxygenase activity by the high
bicarbonate concentrations in the reaction mixtures in the present
experiments. These result from bicarbonate carried over from the
preactivation mixture; a higher bicarbonate concentration is required
to activate the bacterial rubisco fully (30). Additionally, the
relative luminescence yield of R. rubrum rubisco is lower
than that of the spinach enzyme (30). The burst of luminescence in the
initial seconds of catalysis (a further distinguishing feature of
chemiluminescence from R. rubrum rubisco (2)) will have
decayed before measurement of the first spectrum (commencing 5 s
after addition of RuBP) and thus had little influence (Fig.
3c).
How Does Mn2+ Become Excited?--
The luminescence
emitted by Mn2+-activated rubisco offers a unique window
into the oxygenase chemistry catalyzed by this enzyme. One possible
mechanism that would cause excitation of the active-site Mn2+ during oxygenation is an electron-exchange process
involving a transient Mn3+ intermediate (Fig.
5). This mechanism entails a succession
of single-electron transfers initiated by donation of a ground-state electron from Mn2+ to O2 and completed by
acceptance of an electron from the sugar substrate into a higher energy
orbital of Mn3+. This single-electron process would
facilitate the spin inversion required for ground-state, triplet
O2 to react with singlet RuBP while maintaining spin
conservation overall. The free energy change associated with the
strongly exergonic oxygenation reaction, estimated by microcalorimetry
to be ~300 kJ mol
1 (31), is sufficient to achieve the
excitation, even allowing for a somewhat greater energy requirement for
the excitation than that indicated by the wavelength of the radiation
accompanying the decay of the excited Mn2+ to ground state
(151 kJ mol
1 for 800 nm photons).

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Fig. 5.
One possible electron-exchange mechanism for
excitation of the Mn2+ ion during addition of triplet
O2 to the singlet enediolate of RuBP.
R1 = -CH2OPO ;
R2 = -CHOH-CH2OPO .
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This mechanism implies that excitation of the Mn2+ occurs
on every oxygenation turnover, consistent with our previous
observations that the chemiluminescence intensity is always
proportional to the O2 consumption rate (3) and that the
quantum efficiency is extremely low because of competing non-radiative
decay processes (2). Single-electron exchanges are not possible with
the natural metal activator, Mg2+; hence the mechanism of
O2 addition catalyzed by Mg2+-activated rubisco
must be different, involving less facile two-electron processes. This
is consistent with the ~10-fold greater catalytic effectiveness
(kcat/Km for O2)
of the Mn2+-activated oxygenase, compared with its
Mg2+-activated counterpart (32).
The proposed mechanism also implies that Mn2+ excitation
and luminescence is linked to the catalytic step in which the
3'-keto-2'-peroxyarabinitol-P2 intermediate is formed.
Therefore, the hydration and cleavage of this intermediate to form the
oxygenase products, P-glycolate and P-glycerate, should not emit light.
This prediction could be checked by feeding the peroxyketone
intermediate as substrate.
The Physiological Importance of Activation of Rubisco by
Mg2+--
Rubisco-catalyzed oxygenation is maladaptive,
increasing the energy requirements of photosynthesis (33). The
oxygenating capacity of Mn2+-activated rubisco is so large
that photosynthesis supported by this catalyst would be incapable of
sustaining a positive carbon balance in the current Earth atmosphere.
Calculations (34) using a leaf-photosynthesis model (18) and the
kinetic parameters of higher plant manganese-rubisco (32), estimate
that, if Mn2+ replaced Mg2+ in the active site
of rubisco, the CO2 compensation point of the leaf (the
minimum CO2 partial pressure required for positive net
CO2 assimilation) would rise 30-fold to ~1500 µbar.
Natural selection of carboxylation chemistry facilitated by the ligand field of Mg2+, rather than Mn2+ (which has a
vital role in other photosynthetic complexes, such as Photosystem II),
may thus be viewed as one of the enabling fundamentals of autotrophic life.