Department of Biochemistry, Uppsala University, Biomedical Center, Box 576, S-751 23 Uppsala, Sweden
Received for publication, December 15, 2000, and in revised form, January 22, 2001
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ABSTRACT |
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Reduction of dioxygen to water is a key process
in aerobic life, but atomic details of this reaction have been elusive
because of difficulties in observing active oxygen intermediates by
crystallography. Cytochrome cd1 is a
bifunctional enzyme, capable of catalyzing the one-electron reduction
of nitrite to nitric oxide, and the four-electron reduction of dioxygen
to water. The latter is a cytochrome oxidase reaction. Here we describe
the structure of an active dioxygen species in the enzyme captured by
cryo-trapping. The productive binding mode of dioxygen in the active
site is very similar to that of nitrite and suggests that the catalytic mechanisms of oxygen reduction and nitrite reduction are closely related. This finding has implications to the understanding of the
evolution of oxygen-reducing enzymes. Comparison of the dioxygen complex to complexes of cytochrome cd1 with
stable diatomic ligands shows that nitric oxide and cyanide bind in a
similar bent conformation to the iron as dioxygen whereas carbon
monoxide forms a linear complex. The significance of these differences
is discussed.
The four-electron reduction of dioxygen to water is the most
exothermic, non-photochemical reaction available to biology. The energy
liberated in this reaction can be used to activate inert bonds in
difficult synthetic and degradative reactions, or to drive proton pumps
to create transmembrane proton gradients in aerobic respiration. An
understanding of the molecular mechanism of oxygen reduction to water
has thus implications to the understanding of a range of key biological
processes. The aim of this study was to capture an early reaction
intermediate in the oxygen reduction pathway of cytochrome
cd1.
Cytochrome cd1 was first characterized as a
soluble cytochrome oxidase (1, 2). Recent crystal structures of the
dimeric enzyme from Paracoccus pantotrophus (3, 4) show that
each polypeptide chain is divided into two domains. Residues 1-137 form a cytochrome c-like domain, containing a covalently
bound c heme, and residues 138-567 form an eight-bladed
Oxygen reduction by crystalline cytochrome cd1
is a relatively fast process, and strategies worked out earlier for
capturing reaction intermediates during nitrite reduction (5) could not be adopted directly. At room temperature and at atmospheric oxygen pressures, reaction intermediates did not reach high concentrations in
the crystal, and the enzyme was prone to reduction by x-rays during
data collection. Capturing elusive intermediates in the cytochrome
oxidase reaction required other approaches in cryo-trapping and in data collection.
Crystallization--
Cytochrome cd1 from
P. pantotrophus was purified (9) and crystallized (10) as
described previously.
Reaction Initiation and Cryo-trapping--
Crystals were reduced
with 20 mM dithionite in a synthetic mother liquor (2.5 M ammonium sulfate, 50 mM potassium phosphate, pH 7.0). Reduction of crystals took place inside a glove box containing less then 1 ppm oxygen (Belle Technology, Bournemouth, UK). After reduction, excess dithionite was removed by soaking the crystals in
18% glycerol, 2.5 M ammonium sulfate, 50 mM
potassium phosphate, pH 7.0. This solution freezes below Data Collection and Structure Refinement--
Data were
collected on frozen crystals at 100 K on beam lines ID14 EH2 and ID14
EH3 at ESRF,1 Grenoble using
monochromatic x-rays (wavelength 0.98 Å and 0.935 Å, respectively).
Data were processed with Denzo and scaled with Scalepack (12). For
statistics see Table I. A hybrid structure consisting of the N-terminal
domain of the structure of oxidized P. pantotrophus
cytochrome cd1 (1QKS) combined with the
C-terminal part of subunit A and the entire B subunit from the reduced
structure (1AOF) was used for initial phasing of data sets 1 and 2 (see Table I). For initial phasing of data set 3 the structure of oxidized
cytochrome cd1 was used (3, 4). The phases were refined using the simulated annealing algorithm in the CNS
program (13) in combination with programs from the CCP4 suite
(14). Model building was done with O (15).
Trapping Reaction Intermediates--
Prior to exposure to oxygen,
crystals of the oxidized enzyme were reduced with dithionite under
anaerobic conditions in a glove box (see "Experimental
Procedures"). The reduced crystals were transferred to a synthetic
mother liquor solution that also contained 18% glycerol as a
cryoprotectant. This solution could be cooled to about
Spectra of flash frozen crystals remained unchanged at 80 K over
several months. However, x-ray data collection on reactive oxygen
intermediates is not a trivial task because electrons liberated by
x-ray photons during data collection can reduce heme centra even at
liquid nitrogen temperatures (Fig. 1, see also Ref.
17).2
Data Collection Strategy--
A consideration of the elastic,
inelastic, and photoelectric cross-sections of atoms present in protein
crystals suggests that an average of 20 electrons are liberated in the
sample through photoelectric and Auger emissions for each elastically
scattered photon when a beam of 12 keV x-rays (~1 Å wavelength) is
scattered from a protein crystal (18, 19). Photoelectrons ejected this way can travel long distances, spanning several unit cells in a protein
crystal, before reaching thermal equilibrium and further electrons may
be ejected during this process. Thermalized electrons can be captured
by electron-depleted atoms/ions within the crystal. Effective electron
traps in cytochrome cd1 crystals are the
oxidized metal centra and the active oxygen intermediates that may be
present in the active site at the start of data collection. In
addition, redox enzymes have evolved to funnel "loose" electrons
effectively into an oxidized active site.
Fig. 1, e and f, shows spectral changes
associated with the x-ray-induced reduction of the c and
d1 heme irons in crystalline cytochrome
cd1. The process can readily be monitored by
single-crystal microspectrophotometry. The data show that at low x-ray
doses, the redox state of the heme centra was only slightly affected (Fig. 1e), but at high x-ray doses (at doses required for
the collection of a full diffraction data set), a full reduction of the
metal centra took place (Fig. 1f). To minimize x-ray-induced reduction, data were collected on several crystals (11 total), each
with a different starting angle, and a composite data set was later
assembled from the first 10° of data from each crystal. For data
collection and refinement statistics see Table
I.
The Bound Dioxygen Species on the d1 Heme--
Using
techniques described above, an early intermediate, corresponding to a
bound dioxygen species in the cytochrome oxidase reaction of the
enzyme, could be captured (2-min incubation in 15 bar oxygen at
The Oxidation State of the d1 Heme in the
Complex--
In the fully oxidized resting enzyme (3, 4), the
porphyrin ring of the d1 heme is nearly planar.
In the reduced enzyme (5), the d1 heme is
puckered. This feature can be used as an internal indicator for the
oxidation state of the d1 heme. Upon reduction
of cytochrome cd1 in the crystal, Tyr-25, which
ligates the d1 heme in the resting oxidized
enzyme, is released to allow substrate binding (5). Concomitantly, the
c domain refolds resulting in a change in the c
heme coordination from His-17/His-69 (in the oxidized resting enzyme)
to His-69/Met-106 (in the fully reduced enzyme) (5, 8). The axial
ligation of the heme centers can be used as additional indicators for
assessing the oxidation state of the system.
Fig. 2A shows the heme plane puckering in subunit B of the
enzyme. On the proximal side of the d1 heme, the
acetylate groups move toward the porphyrin ring. This movement is
accompanied with a rearrangement of neighboring protein side chains and
solvent molecules. The c heme of this subunit has His/Met
ligation as in the fully reduced enzyme. These features suggest that
both the c and the d1 hemes of
subunit B are reduced. In this case, the bound dioxygen species is most
likely a neutral dioxygen molecule that has not yet been converted to
other dioxygen species (e.g. superoxide or peroxide) by the
enzyme. Further support for this conclusion is the Fe-O-O bond angle of
134° in the complex. We note that electron transfer is usually faster
than heme-religation, and therefore, a certain possibility exists that
the actual oxidation state of these metal centers may differ from what
they seem to be from the structure (7, 8, 20).
Single crystal microspectrophotometry indicates a mixture of oxidation
states for the heme centers in the crystal. The two chemically
identical subunits (A and B) experience different crystallographic environments in the monoclinic crystal (the asymmetric unit contains the entire dimeric enzyme), and the two subunits have different reactivities in the crystal (5). This seems to be the case in the
present study also. The structure of subunit A has His/His axial
ligands for the c heme, and sports a planar
d1 heme on which density for Tyr-25 is connected
to the d1 heme iron (not shown). This suggests
that the A subunit is fully oxidized in the complex and has probably
completed its catalytic cycle before the sample was flash frozen.
The data collection strategy described above allowed us to visualize an
early intermediate in the reduction of dioxygen by cytochrome
cd1. For a comparison, Fig. 2B shows
what happens if 100° of x-ray data are collected from one of the
crystals used to assemble the composite data set from which the map in
Fig. 2A was calculated (see also Fig. 1,
d-f). The 1.6 Å structure (Protein Data Bank
accession number 1HJ4) shows that the dioxygen complex present in the
crystal early in data collection (Fig. 2A) has broken down
during 100° of x-ray exposure. The electron density map of Fig.
2B reflects a mixture of structures, including the original
dioxygen species, a putative mono-oxygen species, and probably also a
water/hydroxyl ion above the heme. The results indicate that
small-scale movements of ligands and side chains may occur during x-ray
data collection even at liquid nitrogen temperatures (see Refs. 21 and
22); however, the frozen state prevents larger structural
rearrangements of the protein in the crystal.
Full Reduction of Dioxygen to Water in the Crystal--
The
dioxygen species captured in Fig. 2A turns over in the
crystal both at 100 K (during data collection) and at 256 K (during prolonged incubation in an oxygen atmosphere). Fig. 2C shows
the 1.46 Å structure (Protein Data Bank accession number 1HJ5) of the
active site of a crystal, which was exposed for 60 min to 15 bar
pressure of dioxygen at Functional Implications--
Oxygen is a highly toxic chemical,
and when it appeared in the atmosphere a couple of billion years ago,
organisms that could convert dioxygen to less harmful compounds had an
evolutionary advantage. A link between oxygen-reducing enzymes and
denitrifying enzymes has been proposed (23, 24), suggesting that higher cytochrome oxidases have evolved by "tinkering with denitrifying enzymes" (25). The present study establishes that the productive binding of dioxygen (Fig. 2A) in the active site is very
similar to the productive binding of nitrite (5), and suggests that the
catalytic mechanisms of oxygen reduction and nitrite reduction are
closely related.
Another aspect concerns the role for heme ligand switching in the
function of cytochrome cd1. A depletion of
reducing equivalents may arise in the periplasm at high oxygen
concentrations. Under those conditions dioxygen or peroxides may react
with an unprotected d1 heme iron and could
create harmful radical species. Shutting off access to the
d1 heme by a return to a resting form in which the d1 heme is shielded prevents the heme iron
from reacting with dioxygen in an uncontrolled manner. If, however,
there is a good supply of external reducing equivalents, the active
site stays open, and dioxygen is reduced to water through the
cytochrome c oxidase activity of the enzyme (8).
Several structures of complexes of P. pantotrophus
cytochrome cd1 with diatomic ligands have been
determined. Fig. 3 shows a comparison of
the dioxygen complex described here and the recently published
structures of the reduced enzyme complexed with CO (16) and
CN
Structural results presented here provide insight into oxygen chemistry
in cytochrome cd1 and on heme centra in general.
The methods described in this paper are also applicable to other
systems including higher oxidases.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
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-propeller domain containing a noncovalently bound
d1 heme, which is the site of oxygen and nitrite
reduction. The role of the c heme is to shuttle electrons
from periplasmic electron donors such as pseudoazurin and cytochrome
c551 to the d1 heme in
the active site. Previous work (5) has shown that the enzyme undergoes major structural rearrangements upon reduction, and produced structures for trapped reaction intermediates in the nitrite reduction reaction of
the enzyme. Based on these crystal structures, a quantum mechanical interpretation of nitrite reduction was proposed (6). It has been
suggested (5, 6) that the structure of the fully oxidized enzyme (3,
4), in which the c and d1 hemes show
His-17/His-69 and Tyr-25/His-200 ligation, respectively, represents a
resting state to which the enzyme does not necessarily return in each catalytic cycle but only when the supply of reducing equivalents is
low. Recent solution studies by EPR and absorption spectroscopy (7)
confirm these speculations, and show the formation of a catalytically
competent form of the enzyme with "switched" c heme axial ligands (His-69/Met-106). Following complete oxidation, this
structure eventually returns to the oxidized resting state. NMR studies
on the isolated c domain (8) are in agreement with this suggestion.
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
25 °C.
Reduced crystals were transferred to a pressure cell (4DX Systems,
Uppsala, Sweden) at
17 °C, and the oxygen pressure was raised to
15 bar. Equipment used was pre-cooled to about
20 °C temperature.
After various times under pressure, the crystals were quickly (<5 s)
frozen in liquid nitrogen. Frozen crystals could be stored for several months without measurable change in their spectra. Spectra were recorded at 100 K with a microspectrophotometer (4DX Systems) (11)
supplied with a cold nitrogen stream (Oxford CryoSystems, Oxford, UK).
RESULTS AND DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
25 °C
before freezing. To slow down oxygen reduction by the crystalline
enzyme and to assure the build up of a relatively high concentration of
oxygen in the crystal, experiments were performed at subzero
temperatures (at
17 °C, where the mother liquor surrounding the
crystal was still fluid) and at elevated oxygen pressures (15 bar).
Plunging crystals exposed to oxygen into liquid nitrogen preserved
potential intermediates in the cytochrome oxidase reaction. The
oxidation state of cytochrome cd1 in the
freeze-quenched crystals was checked by single crystal microspectrophotometry at liquid nitrogen temperatures (Fig.
1). The two chemically identical subunits
experience different crystallographic environments and have been
reported to react at different rates in the crystal (5, 16). Therefore,
crystal spectra were only used as first estimates of how far the
reaction had proceeded in the crystal. Under the experimental
conditions described here, oxygen reduction took several minutes, and
an hour-long incubation of a reduced crystal with oxygen at
17 °C
returned the system to the fully oxidized resting state as judged by
single crystal microspectrophotometry (Fig. 1, a and
c) and x-ray crystallography. Quenching the reaction after 2 min of exposure to oxygen (Fig. 1d) could trap an early
intermediate.
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Fig. 1.
Spectra of frozen single crystals of
cytochrome cd1 nitrite reductase. All
spectra were recorded at 100 K. Spectral features for the c
and d1 hemes are indicated. Variations in the
base lines are due to the different geometries, sizes, and orientations
of the crystals (raw data are shown). a, fully reduced
enzyme; b, fully oxidized enzyme; c, reduced
crystal incubated with oxygen under 15 bar pressure at 17 °C for
60 min; d, reduced crystal incubated with oxygen under 15 bar pressure at
17 °C for 2 min; e, spectrum following
10° of x-ray data collection from d with 0.98 Å x-rays at
beam line ID14 EH2 of the ESRF; f, spectrum after 100° of
data collection from d. Arrows highlight key
spectral features for the d1 heme.
X-ray data collection and refinement statistics
17 °C) and observed in the crystal at 1.6 Å resolution (Fig.
2A). The omit map calculated
from the composite data set using the refined structure (Protein Data
Bank accession number 1HJ3) with the dioxygen molecule removed for
phase calculation shows well resolved density for the bound dioxygen
species. This density extends from the d1 heme
toward the catalytically important His-388 and His-345 residues (3, 5,
6). An unrestrained refinement of the enzyme-dioxygen complex suggests
that the Fe-O distance is 1.8 Å with an Fe-O-O bond angle at 134°.
The distal oxygen atom of the bound dioxygen species forms hydrogen
bonds with His-388 and His-345. The structure also shows a strong
puckering of the d1 heme in the dioxygen
complex.
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Fig. 2.
The bound dioxygen species on the
d1 heme in the active site of cytochrome
cd1. Reduced crystals of cytochrome
cd1 were incubated with oxygen under 15 bar
pressure at 17 °C in a cryoprotectant solution. After 2 min in the
oxygen atmosphere, crystals were flash frozen in liquid nitrogen
(see "Experimental Procedures"). X-ray-induced reduction of the
bound dioxygen species (see text) was minimized collecting 10° of
data from each of eleven differently oriented crystals and were merged
into a unique composite data set (data set 1 in Table I). The structure
in A shows the dioxygen species in the cytochrome oxidase
reaction of the enzyme, whereas B shows the same structure
after 100° of x-ray data had been collected from one single crystal.
In this case, many electrons were liberated in the sample during data
collection, and these electrons have driven the reaction further.
C, the structure when the crystal was exposed for 60 min to
dioxygen at 15 bar pressure at
17 °C before flash freezing. This
structure shows the rebinding of Tyr-25 to the
d1 heme iron at the end of the reaction as the
enzyme returned to a fully oxidized resting state (Fig. 1b).
The three-dimensional arrangement of Asp-346, His-388, and the distal
oxygen atom of the bound dioxygen species in A resembles the
Asp-His-Ser-OH catalytic triad in serine proteases. The electron
density shown in A and C are "omit" maps
calculated as 2Fobs-Fcalc
maps after omitting the dioxygen molecule (A) or the Tyr-25
side chain (C). The electron density shown in B
is a 2Fobs-Fcalc map.
Maps are contoured at 1.4
(A and B) or 1.2
(C) where
is the root mean square electron
density for the unit cell.
17 °C (see also Fig. 1c), and then flash frozen in liquid nitrogen for data collection. The originally oxidized iron centra (Fig. 1c) in the crystal
became reduced during data collection (not shown). However, the glass state of the surrounding solution prevents the large structural rearrangements (characteristic for the reduced enzyme) at 100 K in the
crystal; the structure shows His-17/His-69 ligation for the
c heme (characteristic for the resting oxidized c
heme) and Tyr-25/His-200 for the d1 heme
(characteristic for the resting oxidized
d1 heme). These findings suggest that at the end
of the catalytic cycle, the enzyme returned to a fully oxidized resting state (Fig. 1c) before it was flash frozen in liquid
nitrogen. These results demonstrate that the crystalline enzyme can
complete a catalytic cycle in the cytochrome oxidase reaction at
17 °C and is capable of returning to a fully oxidized resting
state at the end of this reaction before flash freezing in liquid nitrogen.
(26). A structure of an NO complex obtained by soaking
a reduced crystal in nitrite (5) is also shown. A recent paper (16) shows that the binding of the neutral carbon monoxide molecule to the
active site of cytochrome cd1 is accompanied by
the rapid release of a proton from the enzyme. This process is
reversible, and the release of the bound CO molecule from the
d1 heme is followed by the uptake of a proton
from bulk solvent. This is a curious phenomenon, which may also
accompany the binding of other neutral diatomic ligands such as
dioxygen to the active site. If the routes for proton uptake and proton
release are different, this could introduce vectoriality into the
system. There are, however, certain differences in the binding
geometries of the various ligands. Compared with CO and
CN
, the dioxygen ligand is bound in a highly bent
conformation. This is consistent with the increased
character given
by the larger number of electrons in the oxygen molecule. The distal atom in three of the diatomic ligands (dioxygen, nitric oxide, and
cyanide) is coordinated by His-388 and His-345, whereas CO is bent
toward a more hydrophobic part of the active site pocket and the oxygen
atom of CO is linked to His-388 only. This conformation is also adopted
by NO in an alternative structure (Refs. 5 and 6, and not shown) where
the oxygen position of NO seen in Fig. 3C between His-388
and His-345 is taken up by the side chain of Tyr-25. The existence of
dual conformations for NO is thought to be important for effective
product release (5, 6).
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Fig. 3.
Comparison of known complexes of cytochrome
cd1 from P. pantotrophus
with diatomic ligands. A, the dioxygen complex at
1.6 Å resolution as described in this study (Protein Data Bank
accession number 1HJ3, subunit B). B, reduced enzyme
complexed with CO at 1.57 Å resolution (Protein Data Bank accession
number 1DY7, subunit B) (16). C, reduced enzyme complexed
with cyanide at 1.59 Å resolution (Protein Data Bank accession number
1E2R, subunit B) (26). D, NO complex at 1.8 Å resolution on
an oxidized d1 heme obtained by soaking a
reduced crystal in nitrite solution (Protein Data Bank accession number
1A0Q, subunit B) (5).
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ACKNOWLEDGEMENTS |
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We thank Drs. E. H. J. Gordon, M. Svensson-Ek, and E. Steensma for critical reading of the manuscript, and Prof. S. J. Ferguson for discussion. We are grateful to MAX-lab, ESRF, and EMBL for beam time allocation. Special thanks to Ed Mitchell for help.
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FOOTNOTES |
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* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The atomic coordinates and structure factors (codes 1HJ3, 1HJ4 and 1HJ5 ) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).
To whom correspondence should be addressed: Department of
Biochemistry, Box 576, S-751 23 Uppsala, Sweden. Tel.: 46-18-4714449; Fax: 46-18-511755; E-mail: janos@xray.bmc.uu.se.
Published, JBC Papers in Press, January 26, 2001, DOI 10.1074/jbc.M011312200
2 G. Carlsson, A. Bergluord, and J. Hajdu, unpublished data.
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ABBREVIATIONS |
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The abbreviations used are: ESRF, European Synchrotron Radiation Facility; NO, nitric oxide; CO, carbon monoxide.
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