JOURNAL OF STRUCTURAL BIOLOGY
ARTICLE NO. 0076
117, 117–123 (1996)
Two-Dimensional Structure of Membrane-Bound Nitrite Oxidoreductase
from Nitrobacter hamburgensis
E. SPIECK1
Department of Microbiology, Institute for General Botany, University of Hamburg, Ohnhorststr. 18, D-22609 Hamburg, Germany
S. MÜLLER
AND
A. ENGEL
M. E. Müller-Institute for Microscopic Structural Biology at the Biozentrum, University of Basel, CH-4056 Basel, Switzerland
E. MANDELKOW
AND
H. PATEL2
Max-Planck-Unit for Structural Molecular Biology, c/o DESY, Notkestr. 85, D-22607 Hamburg, Germany
AND
E. BOCK
Department of Microbiology, Institute for General Botany, University of Hamburg, Ohnhorststr. 18, D-22609 Hamburg, Germany
Received December 20, 1995, and in revised form April 18, 1996
from the oxidation of nitrite to nitrate under aerobic
conditions. The organisms can also grow anaerobically by dissimilatoric nitrate reduction (Freitag et
al., 1987). The lithotrophic growth of Nitrobacter is
slow and inefficient, since nitrite oxidation is thermodynamically unfavorable (Cobley, 1976). The primary energy product was shown to be NADH (Sundermeyer and Bock, 1981a), which is used for ATP
synthesis (Freitag and Bock, 1990). Up to now it has
not been clear how energy conservation occurs.
Similar to phototrophic bacteria, cells of Nitrobacter possess an extensive intracytoplasmic membrane
system. The key enzyme of nitrite oxidation—the
nitrite oxidoreductase (NOR)—is organized in 7- to
10-nm particles, which are densely packed on the
inner side of the cytoplasmic and intracytoplasmic
membranes (Tsien et al., 1968; Remsen and Watson,
1972; Sundermeyer and Bock, 1981b; SundermeyerKlinger et al., 1984). Accordingly, electron microscopy of the isolated enzyme has revealed uniform
particles with a size of 8 nm (Meincke et al., 1992). In
addition, particulate membranes were labeled with
monoclonal antibodies, recognizing the a- and b-subunits of NOR (Spieck et al., 1996). The concentration
of NOR in cells of Nitrobacter varies with growth
conditions. It is the major constituent of nitrite
oxidizing membranes, representing 10–30% of the
total protein (Tanaka et al., 1983; Bock et al., 1991).
Quasi-crystalline arrays of NOR particles are vis-
Isolated membranes of the facultative nitrite oxidizing bacterium Nitrobacter hamburgensis X 14
displayed a periodic arrangement of the membranebound nitrite oxidoreductase (NOR). The crystallinity of these two-dimensional NOR arrays was improved by polyethylene glycol treatment. Negative
stain electron microscopy and digital image processing were used to analyze the structure of NOR. The
lattice vectors had a length of 9.7 6 0.4 and 11.8 6 0.4
nm, including an angle of a 5 71°. Diffraction patterns of the oblique lattice extended to the third
order indicating a resolution of D2.9 nm. The correlation averaged projection suggested a twofold symmetric unit cell composed of two enzyme particles
with an asymmetric shape, showing a larger and a
smaller morphological domain. The molecular
weight of a single NOR particle was found to be
186 6 43 kDa by scanning transmission electron
microscopy, suggesting that this particle is an
ab-heterodimer. r 1996 Academic Press, Inc.
INTRODUCTION
The facultative lithoautotroph Nitrobacter hamburgensis X 14 (Bock et al., 1983) derives its energy
1 To whom correspondence should be addressed. Fax: 040/82282431.
2 Present address: Purdue University, West Lafayette, IN 47907.
117
1047-8477/96 $18.00
Copyright r 1996 by Academic Press, Inc.
All rights of reproduction in any form reserved.
118
SPIECK ET AL.
ible on electron micrographs of isolated membranes
by freeze etching or negative stain electron microscopy (Bock and Heinrich, 1971; Remsen and Watson,
1972). In ultrathin sections particles exhibited electron-dense layers associated with the inner membrane surfaces (Murray and Watson, 1965; Remsen
and Watson, 1972).
Depending on the isolation procedure, the monomeric NOR consists of 2–3 subunits (Tanaka et al.,
1983; Sundermeyer-Klinger et al., 1984; Meincke et
al., 1992; Bock et al., 1991). The membrane-associated a-subunit (115 kDa) and b-subunit (65 kDa) are
solubilized by heat treatment (Meincke et al., 1992).
The isolated ab-complex is catalytically active and
includes molybdenum and iron–sulfur centers (Meincke et al., 1992). In contrast, cytochromes a1 and c1
have been coisolated from native membranes with
NOR using Triton X-100, n-octylglycoside, or Nadeoxycholate (Tanaka et al., 1983; SundermeyerKlinger et al., 1984; Bock et al., 1991). In the latter
case, the integral protein cytochrome c1 was described as the g-subunit (32 kDa) of the enzyme
(Sundermeyer-Klinger et al., 1984). Recently, slight
differences were found in molecular weights of the
a-subunit (130 kDa) and the b-subunit (58 and 62
kDa) (Kirstein and Bock, 1993; Ehrich et al., 1995).
Kirstein und Bock (1993) have identified the genes of
these subunits, but only the b-subunit was sequenced completely. This subunit was shown to be
an electron-channeling protein, acting between the
nitrite-oxidizing a-subunit and the membraneintegrated electron transport chain.
Here we describe the projection of the twodimensional NOR crystals as derived from negatively stained samples by electron microscopy and
digital image processing. The mass of NOR particles
was measured by scanning transmission electron
microscopy, suggesting that the NOR particles of
approximately 8 nm are ab-heterodimers.
MATERIALS AND METHODS
Organism and growth conditions. N. hamburgensis strain X
14 was grown mixotrophically with nitrite and organic matter as
described by Bock et al. (1983).
Isolation of cytoplasmic and intracytoplasmic membranes. Cells
were harvested at the end of the logarithmic growth phase by
centrifugation and suspended in TEMB buffer without EDTA (10
mM Tris–HCl, 10 mM MgCl2, 20 mM NaHCO3, pH 8,0). After
sonication (Branson, 20 KHz, 3 3 2 min) the broken cells were
removed by centrifugation at 8000g for 10 min. Membranes were
isolated from the supernatants by sucrose density gradient centrifugation according to Milde and Bock (1984), suspended in
TEMB buffer, and kept at 220°C until used.
Purification of NOR. The enzyme was isolated from cytoplasmic and intracytoplasmic membranes by heat treatment. Membranes were heated at 55°C for 25 min in TENN buffer (10 mM
Tris–HCl, 1 mM EDTA, 29 mM NaNO2, 20 mM NaHCO3, pH 8,3)
and cooled to 4°C. Purification of NOR was performed by sucrose
density gradient centrifugation as described by Meincke et al. (1992).
Analytical procedures. Protein was determined according to
Bradford (1976) using bovine serum albumin as standard.
Enzyme activities. Nitrite oxidase activity was measured by
the consumption of nitrite with NaClO3 as artificial electron
acceptor (Meincke et al., 1992). One unit was defined as mmol
NO22/min 3 mg protein.
Preparation of crystalline arrays. Membranes were thawed
and diluted with TEMB buffer to protein concentrations of 0.5–1
mg/ml. The following procedures were applied in an attempt to
improve the order of the crystalline arrays.
A. Membranes suspended in TEMB buffer were incubated in
0.5% polyethylene glycol (PEG) 4000 (Merck) for 2 days at 4°C.
B. Membranes were dialyzed against 5% PEG 6000 (Merck) in
TEMB buffer at 4°C for 7 days.
C. Membranes were treated with phospholipase A2 (Sigma) for 2
days at 4°C following the protocol of Mannella (1984).
Electron microscopy and digital image processing. Membranes were negatively stained by a modified method of Valentine
et al. (1968) using uranyl acetate (2%) or sodium phosphotungstate (1%, pH 7.2). Electron microscopy was performed with an
EM 201 and EM 420 (Philips) at 60–80 kV and magnifications of
36 000–49 000. Micrographs were selected for computer analysis
based on their optical diffraction patterns. Selected areas containing up to 300 unit cells were digitized into 256 3 256 pixel squares
by a densitometer (Optronics) using a step size of 25 µm,
corresponding to a sampling distance of 0.7 nm. Initial image
averaging was performed by Fourier peak filtration (for a review
of these methods see Amos et al., 1982). However, lattice distortions necessitated the application of correlation averaging (Saxton and Baumeister, 1982). To this end, a small reference area of
the image was cross-correlated with the whole field, and peak
values and coordinates of the correlation maxima were stored.
Real space averages were then calculated from areas exhibiting a
high correlation with the reference selected. Several refinement
passes taking the previous average as new reference ensured an
unbiased final average. One unit cell was then extracted, its
twofold phase origin determined, and the root mean square
deviation from the p2 symmetry measured.
Mass determination by scanning transmission electron microscopy. First membranes or isolated NOR particles were adsorbed
for 1 min to glow-discharged thin carbon films mounted on grids
covered with a thick fenestrated carbon layer. In a second step
tobacco mosaic virus (TMV) was coadsorbed. Grids were then
washed extensively in quartz bidistilled water. Excess liquid was
removed and the grids plunged into liquid nitrogen and freezedried within the scanning transmission electron microscope
(STEM) (Müller et al., 1992). Dark-field images containing 512 3
512 pixels were recorded from the unstained preparations using a
Vacuum Generators STEM HB-5 at 80 kV, magnifications of
200 000, and recording doses between 300 and 800 e/nm2. The
TMV served as an internal mass standard (Wall and Hainfeld,
1986), allowing scaling for both instrument fluctuations and beam
induced mass loss. The evaluations were made using the program
package IMPSYS (Müller et al., 1992). The particle masses
resulting after integration and background substraction were
presented in histograms.
RESULTS
NOR particles were bound to the inner side of the
cytoplasmic and intracytoplasmic membranes of Nitrobacter, protruding approximately 8 nm from the
membrane surface (Fig. 1a). Electron microscopy of
the isolated enzyme revealed nearly globular to
slightly elongated particles of average size 7.7 3 9.8
nm (Fig. 1b).
After gentle purification of the membranes, large
NITRITE OXIDOREDUCTASE IS A HETERODIMER
FIG. 1. NOR particles of Nitrobacter hamburgensis X 14,
negatively stained with uranyl acetate. (a) Membrane-bound
NOR particles on the cytoplasmic side of a membrane fragment.
The arrowheads mark periodically arranged particles in side view,
protruding approximately 8 nm from the membrane surface. Bar,
50 nm. (b) Sequence of isolated NOR particles. Bar, 20 nm.
fragments (up to 1 µm) with small and poorly
ordered crystalline areas of membrane-bound NOR
were observed. Although mainly single-layered membrane sheets were obtained, sometimes closed envelopes showing moiré patterns were also present (not
shown). Particulate membranes oxidized nitrite to
nitrate with a specific activity of 3.8–4.4 units (not
shown).
To improve the crystallinity of the NOR arrays,
membranes were treated with PEG. Incubation with
0.5% PEG 4000 for 2 days at 4°C resulted in large
crystalline areas (Fig. 2a). The arrangement of the
surface particles in parallel rows was clearly visible.
Dialysis against 5% PEG 6000 for 7 days at 4°C also
resulted in crystals suitable for image analysis (Fig.
2b). Treatment with PEG for 5–7 days resulted in
specific activities of 1.5 to 2.5 units which compared
favorably to controls. The formation of crystalline
areas was observed less frequently after treatment
with phospholipase A2 (Fig. 2c). Comparing the
arrangements of the particle lines in Fig. 2a with
those in Figs. 2b and 2c, two different orientations
were visible, probably due to different sides of the
crystal sheets lying on the grid. The three crystals of
Figs. 2a–2c exhibited unit cell dimensions a 5 9.7 6
0.4 and b 5 11.8 6 0.4 nm, with an angle between the
lattice lines of a 5 71° 6 2°. Two diffraction orders
were observed reproducibly in the optical diffraction
patterns of crystalline areas (not shown). Additional
119
reflections were obtained in computer-calculated diffraction patterns, because the best area could be
selected more easily than with the optical diffractometer. As displayed in Fig. 3, the diffraction pattern of
the area marked in Fig. 2a showed spots up to order
(3,3), indicating a resolution of about 2.9 nm.
The averages in Figs. 2a–2c (insets) calculated
from the amplitude and phase of the Fourier peaks
demonstrate that the NOR was arranged in parallel
rows with close contacts along the y-axis. Average 2a
(inset) was turned over to have the same orientation
as averages 2b (inset) and 2c (inset). All of the
reconstructions exhibit an elongated stain excluding
structure with a length of 10 6 1 nm and a width
which varied from 3 6 0.5 to 5 6 0.5 nm. Most details
are visible in Fig. 2a (inset), where the asymmetric
unit exhibited two domains of different width, but
approximately equal length along the y-axis. The
image reconstruction in Fig. 2b (inset) shows bilobed
structures with equal domains, while in Fig. 2c
(inset) only a weak indication of two domains is visible.
The variability of the averages shown in Figs.
2a–2c (insets) prompted an attempt to unveil the
projection of negatively stained NOR particles by
correlation averaging (Saxton and Baumeister, 1982).
To this end, averages were calculated from the
crystalline arrays shown in Figs. 2a and 2b which
had been obtained by incubation with PEG 4000 or
by dialysis against PEG 6000. The individual correlation averages exhibited root mean square deviations
from p2 symmetry of 4 and 6%, respectively. Hence,
both averages were twofold symmetrized. After turning over the average of the micrograph shown in Fig.
2a, the averages were added to yield the final
projection map displayed in Fig. 4. The unit cell
contained two asymmetric particles, exhibiting a
larger and a smaller morphological domain.
The STEM was used to determine the mass characteristics of particulate membrane sheets and of the
free particles. The raw data were scaled according to
the coadsorbed TMV mass-per-length values, using
the sequence value of 131.5 kDa/nm as reference
(Namba and Stubbs, 1986). This autometically corrected for beam induced mass loss to a first approximation. Freeze-dried membranes yielded a wide
variety of mass-per-area values that could not be
interpreted (not shown). However, free particles of
two different sizes were also visible on the grids (Fig.
5a). The small particles with an elongated to globular shape had an average size of 7.5 3 9.0 nm.
Calculated from their electron scattering power the
average mass of 291 individual particles (n) was
190 6 55 kDa after scaling. The large particles
present in the same preparation had a globular
appearance with a diameter of about 15 nm and a
mass of 1238 6 114 kDa (n 5 214). In a second
experiment mass values of 184 6 31 kDa (n 5 200)
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SPIECK ET AL.
NITRITE OXIDOREDUCTASE IS A HETERODIMER
FIG. 3. Computer-generated diffraction pattern of the crystalline array marked in Fig. 2a. The diffraction spots (3,3) extended
to a resolution of 2.9 nm (arrows). (a*,b*) Lattice vectors.
for the small and 1199 6 92 kDa (n 5 322) for the
large particles were determined (Table I). The combined data from these two membrane preparations is
shown as a histogram in Fig. 5b. Finally, the mass of
NOR which had been dissociated from the membranes by heat treatment was measured. In this
case, the histogram showed peaks at molecular
weights of 682 6 119 kDa (n 5 160) and 1245 6 119
kDa (n 5 34) (Table I). An insignificant number of
particles in the 200-kDa mass range was found. The
two mass peaks correspond to larger diffuse and
well-defined more compact particles, respectively.
DISCUSSION
The membrane-associated NOR of N. hamburgensis X 14 forms quasi-crystalline areas. These nitrite
oxidizing membranes were investigated by electron
microscopy and digital image analysis. The projection map of NOR showed the size, shape, and ultrastructure of the enzyme in its native environment.
Lattice distortions and the small size of crystalline
arrays limited the resolution achieved by Fourier
peak filtration to about 2.9 nm. Therefore, projection
121
FIG. 4. Image reconstruction of the NOR by correlation averaging of two individual electron micrographs (Figs. 2a 1 2b). The
unit cell contained two asymmetric NOR particles. The average
was twofold symmetrized (see text). Protein appears bright. (a1b1 )
Proposed subunit composition of a single NOR particle. (a,b)
Lattice vectors.
maps were calculated by correlation averaging (Saxton and Baumeister, 1982).
The final map of the enzyme obtained by superimposing two individual correlation averages showed
unit cells comprising two asymmetric particles. These
particles could not be resolved in the averages
calculated from the peaks in the Fourier transforms.
The variation of these averages is probably due to
the poor order of the crystals which exhibited lattice
faults and distortions. Correlation averaging techniques allowed unit cells to be extracted that displayed more reproducible features. According to the
final map, the contour level of the stain envelope
surrounds an area of about 60 nm2. As documented
by Fig. 1 the particles protrude by approximately 8
nm from the membrane surface. Taking the mass
density of protein as 0.818 kDa/nm3, we calculate a
mass of 393 kDa per unit cell which corresponds to 2
ab-heterodimers. Such ab-heterotetramers of the
NOR have been described in the literature (Bock and
Heinrich, 1971; Remsen and Watson, 1972). They
FIG. 2. Negatively stained nitrite oxidizing membranes of N. hamburgensis X 14 with crystalline arrays of NOR particles. Marked
areas were taken for image reconstructions. Negative staining with sodium phosphotungstate. Bars, 100 nm. (a) Incubation with PEG 4000
(preparation A). (b) Dialysis against PEG 6000 (preparation B). (c) Treatment with phospholipase A2 (preparation C). (Insets) Image
reconstructions of the NOR by Fourier transformation with definition of the unit cell. Protein appears bright. All averages are shown with
the same handedness. Bars, 5 nm. The lattice spacings of the crystalline arrays were (a) 9.5 3 10.8 nm; (b) 9.1 3 11.5 nm; (c) 8.8 3 11.3 nm.
122
SPIECK ET AL.
FIG. 5. STEM mass determination of NOR. (a) Freeze-dried membrane-depleted NOR particles in STEM dark-field images. The
particle distribution was heterogenous in size. Bar, 100 nm. s, small particle; l, large particle; V, TMV. (b) Mass distribution of NOR
particles from membrane suspensions 1 and 2 (Table I) combined in one histogram. The two peaks correspond to monomers (186 6 43 kDa)
and hexamers (1211 6 101 kDa).
have also been found in fragmented lattices (unpublished results). Thus, they appear to be the basic
structural elements of the crystals and may be the
functional unit of the NOR. This is in agreement
with the a2b2g1-stoichiometry of detergent-solubilized NOR complexes (Sundermeyer-Klinger et al.,
1984). A similar structure was found for the denitrifying bacterium Rhodobacter sphaeroides. For these
organisms Sabaty et al. (1994) described a wellordered dimeric pattern of membrane proteins, when
TABLE I
STEM Mass Determination of Free NOR Particles from
Nitrobacter hamburgensis X 14, Present in Membrane
Suspensions upon Freeze Drying or Obtained by Heat
Treatment of the Membranes
Sample
Membranes 1
Membranes 2
Heat treated
Type of
particle
Calculated
molecular
mass
(kDa) 6SD
Number
of counts
Dose
(e/nm2)
Small
Large
Small
Large
Diffuse
Large
190 6 55
1238 6 114
184 6 31
1199 6 92
682 6 119
1245 6 119
291
214
200
322
160
34
608 6 166
553 6 186
610 6 181
598 6 185
544 6 168
544 6 168
cells were grown phototrophically in the presence of
nitrate.
NOR particles are only loosly associated with the
membrane. Many are lost during isolation of the
membrane crystals or at high ionic strength (Sundermeyer and Bock, 1981b) as documented by Figs. 2a
and 2c in which free particles are evident. For mass
measurements in STEM, specimens are necessarily
unstained. The resulting low contrast did not allow
surface structure to be distinguished on the membranes; consequently, no selection could be made for
uniform crystalline areas. This resulted in a wide
range of mass-per-area values that could not be
interpreted. However, the mass of individual particles could be determined.
The smallest particles with a mass of 186 6 43
kDa occurred in membrane preparations which had
been freeze-dried. In accordance with the data of
Meincke et al. (1992) which showed that the monomeric form of NOR consists of two subunits with
molecular weights of 115 and 65 kDa respectively,
they correspond to ab-heterodimers. The largest
particles with a mass of 1211 6 101 kDa occurred in
all three preparations examined. However, they
formed only a minor population when heat treatment had been employed. Their mass corresponds to
NITRITE OXIDOREDUCTASE IS A HETERODIMER
6 ab-heterodimers, that is, 3 unit cells. Their relatively high occurrence would seem to indicate a
particular stability for such aggregates. The fairly
large standard deviation measured may be explained by the loss of an ab-heterodimer in some
cases. Where heat treatment was employed, dissociation from the membrane was both influenced and
enforced. The main species then found corresponded
to an aggregate of 4 ab-heterodimers rather than 6.
Again, the relatively large standard deviation measured may be explained by the loss of an abheterodimer in some cases. Interestingly essentially
no monomeric ab-heterodimers were detected. This
fact and the low preservation of the membranes
examined in STEM indicates that the membranes
may have lost some monomers during the grid
preparation, washing, and freeze-drying procedure
used. It is reasonable to assume the absence of the
g-subunit in such monomers since extraction of this
is known to require detergent treatment (Tanaka et
al., 1983; Sundermeyer-Klinger et al., 1984; Bock et
al., 1991). Thus, the aggregation state of NOR
ab-heterodimers observed upon removal from the
membrane would seem to depend on the isolation
method employed. The mass determined for the
smallest particles, single ab-heterodimers, agrees
with the unit cell mass calculated assuming a stoichiometry of a2b2 almost exactly.
This paper is based on a Ph.D. thesis of E. Spieck submitted to
the Faculty of Biology, University of Hamburg.
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