(Received for publication, November 27, 1995; and in revised form, January 30, 1996)
From the
The Bradyrhizobium japonicum cbb-type
cytochrome oxidase, which supports microaerobic respiration, is a
multisubunit enzyme encoded by the genes of the fixNOQP operon. We investigated the contribution of the individual
subunits to function and assembly of the membrane-bound complex.
In-frame deletion mutants of fixN, fixO, and fixQ, and an insertion mutant of fixP were
constructed. All mutants, except the fixQ mutant, showed
clearly altered absorption difference spectra of their membranes and
decreased oxidase activities, and they were unable to fix nitrogen
symbiotically. The presence of the individual subunits was assayed by
Western blot analysis, using subunit-specific antibodies, and by heme
staining of the c-type cytochromes FixO and FixP. These
analyses led to the following conclusions: (i) FixN and FixO are
necessary for assembly of the multimeric oxidase, (ii) FixN and FixO
assemble independently of FixP, and (iii) FixQ is not required for
complex formation and, therefore, does not seem to be an essential
subunit. The possible oxidase biogenesis pathway involves the formation
of a primary core complex consisting of FixN and FixO, which allows the
subsequent association with FixP to form the complete enzyme.
Terminal oxidases are the ultimate components of respiratory
chains catalyzing the four-electron reduction of molecular oxygen to
water. The bacterial respiratory system is usually branched and
comprises several terminal oxidases, allowing bacteria to grow at
different oxygen tensions. Most of the bacterial oxidases belong to the
so-called superfamily of heme-copper oxidases which is divided into two
groups, the quinol oxidases and the cytochrome c oxidases (for
a review, see (1) ). Recently, a new type of heme-copper
oxidase with regard to subunit composition and content of prosthetic
groups has been discovered, the cbb-type oxidase.
Genes encoding this oxidase were isolated first from several rhizobial
species (2, 3, 4, 5) and called fixNOQP as they supported symbiotic nitrogen fixation.
Meanwhile, homologous genes have also been identified in other
Gram-negative bacteria(6, 7) . The cbb
-type oxidase has been purified from several
species and characterized
biochemically(8, 9, 10, 11, 12) .
It acts as a cytochrome oxidase, not as a quinol oxidase, and contains
three major subunits: a membrane-integral b-type cytochrome
(subunit I) with a low-spin heme and a high-spin heme-Cu
binuclear center, and two membrane-anchored c-type
cytochromes. Interestingly, there is no Cu
-containing
subunit, in contrast to conventional cytochrome c oxidases(8, 9) .
In Bradyrhizobium
japonicum, the fixNOQP-encoded cbb-type oxidase is expressed only under
microaerobic and anaerobic conditions(2) . The 61-kDa fixN gene product has up to 14 putative transmembrane helices and binds
heme B(2, 11) . The fixO and fixP genes code for membrane-anchored c-type cytochromes of 28
and 32 kDa, respectively. FixO is a monoheme cytochrome c and
FixP probably a diheme cytochrome c. FixQ is a 54-amino-acid
polypeptide that is thought to be membrane-bound by its hydrophobic
N-terminal half. Having a K
for O
as low as 7 nM, the B. japonicum
cbb
-type oxidase supports respiration under
oxygen-limiting conditions and is, therefore, required for nitrogen
fixation in symbiosis with soybean (Glycine max L.
Merr)(2, 11) .
Correct folding and assembly of the
individual subunits of a multimeric membrane protein complex may
involve a specific nucleation and a precise pathway. Little is known
about the assembly of oxidase complexes in bacteria. Minagawa et
al.(13) reported the absence of the Escherichia coli
cyoABCD gene products when only one of the subunit genes was
deleted. Studies on assembly of the Paracoccus denitrificans
aa-type oxidase revealed that subunit I was not
present in a subunit II gene deletion mutant, whereas subunit III was
not absolutely essential for the formation of a subunit I
II
subcomplex(14, 15) . Using the B. japonicum
cbb
-type oxidase system as an example, we attempted to
elucidate the role of its individual subunits in assembly and
biological function.
Figure 1:
Physical map of the B. japonicum
fixNOQP region and of mutant constructs. A, the top
line shows a restriction map of the DNA region harboring the 3`
part of orf277, orf141, fixNOQP and the 5`
part of fixG. Restriction sites: Bb, BbsI; Bg, BglII; Bs, BsaI; E, EcoRV; Ec, Eco47III; Nc, NcoI; Ns, NsiI; P, PvuII; Ps, PstI; R, EcoRI; S, SacII. B, plasmids used for marker replacement
(double crossing-over) in the chromosome. The dashed line represents a deletion, and the filled arrowhead the
transcriptional orientation of the inserted aphII cassette. C, DNA fragments used to complement Bj4503
(fixNOQP) by cointegration (single cross-over). Deletions
are represented by the
.
All of the newly fused sites at which remote DNA was joined after in-frame deletion were confirmed by sequencing. The plasmids pRJ4526, pRJ4518, and pRJ4528 were cointegrated into the Bj4503 chromosome by homologous recombination. Tetracycline-resistant clones were selected (single crossing over) and their DNA examined by Southern blot hybridization for the correct genomic structure. In the case of Bj4526, plasmid cointegration occurred at the fixG locus, i.e. downstream of the operon deletion, whereas the plasmids pRJ4518 and pRJ4528 cointegrated within the orf277orf141 locus, i.e. upstream of the operon deletion.
All fusion sites were confirmed by sequencing. While pRJ3603 was cointegrated into the chromosome of the wild type B. japonicum 110spc4 at the fixNOQ locus, the plasmids pRJ4538, pRJ4539, pRJ4540, pRJ4542, pRJ4543, pRJ4545, pRJ4550, and pRJ4569 were cointegrated into the operon deletion mutant Bj4503 by conjugation. Tetracycline-resistant clones were selected and confirmed by Southern blot hybridization. Thus, strains Bj4538, Bj4539, Bj4540, Bj4542, Bj4543, Bj4545, Bj4550, and Bj4569 were obtained that contain the corresponding plasmids integrated at the homologous position in the chromosome.
Figure 2:
Western blot analysis of membrane
proteins. Four similar blots were developed using immunoglobulins
against FixN, FixO, FixP and cytochrome c, as
indicated on the right. Membranes were prepared from
microaerobically grown cells. Approximately 6-10 µg of
membrane protein were loaded in each lane. The following strains were
tested: Bj3067 (bc
mutant, fbcF::
, lane 1), 110spc4 (wild type, lane 2), Bj4503 (
fixNOQP, lane 3),
Bj4504 (fixNOQP
by complementation, lane
4), Bj4526 (
fixN, lane 5), Bj4518
(
fixO, lane 6), Bj4528 (
fixQ, lane 7), and Bj3618 (fixP::aphII, lane
8).
The membrane protein
fractions of the same strains were also tested for heme C incorporation
into the FixO or FixP proteins. Proteins were separated by SDS-PAGE and
stained for covalently bound heme. Again, the complemented strain
served as a positive control. Heme staining revealed four
membrane-bound cytochromes c (Fig. 3, lanes 2, 4, and 7): a strongly staining 32-kDa protein that
had been identified previously as FixP, the diheme cytochrome c(2) and which was also seen in the bc mutant (lane 1); the
20-kDa CycM protein (32, 34) that was present in all
of the strains (lanes 3, 5, 6, and 8 included), except in the bc
mutant (lane 1); the 28-kDa FixO protein (lanes
1, 2, 4, 7, and 8; the lower
of two bands) which almost comigrates with cytochrome c
(lanes 2-8; the upper of the two bands). A
comparison between the Western blot (Fig. 2) and heme staining
analyses (Fig. 3) clearly showed that the upper band of the
28-kDa double band must be attributed to cytochrome c
, whereas the lower one represents FixO. This
finding is corroborated by the fact that the 28-kDa heme staining
double band was completely missing in a bc
/fixO
double mutant (data not shown). The presence or absence of FixO
and FixP in heme stains completely concurred with their presence or
absence in Western blots. Hence, heme C is covalently bound to FixO and
FixP in the complemented strain, the bc
mutant, and the
fixQ mutant; in addition, FixO is also detectable in the fixP::aphII mutant.
Figure 3:
Analysis of c-type cytochromes by
heme staining. Membrane proteins were separated by SDS-PAGE and stained
for covalently bound heme. Microaerobically grown cells from the
following strains were tested: Bj3067 (fbcF::, bc
, lane 1), 110spc4
(wild type, lane 2), Bj4503 (
fixNOQP, lane
3), Bj4504 (fixNOQP
by complementation, lane 4), Bj4526 (
fixN, lane 5), Bj4518
(
fixO, lane 6), Bj4528 (
fixQ, lane 7), and Bj3618 (fixP::aphII, lane 8). Lanes 1, 3, 5, 6, and 8 contained approximately 200 µg, the other lanes 100 µg of
protein. In the operon deletion and in the
fixN,
fixO, and fixP::aphII mutants, a new, weakly
heme-staining band of unknown identity appeared at approximately 26
kDa. The apparent molecular masses of the proteins (kDa) are shown on
the right.
The presence of heme B in the oxidase
was investigated spectroscopically. Solubilized membrane fractions were
used in these experiments. As shown in previous work(11) ,
peaks characteristic for cytochrome b at 558 nm and for
cytochrome c at 552 nm were detected in wild type membranes of
microaerobically grown cells. The interpretation of dithionite-reduced minus air-oxidized difference spectra of membranes is somewhat
complicated by the fact that a number of different b- and c-type cytochromes are present whose absorption maxima overlap
in the range of 550 to 560 nm. The spectrum of the operon deletion
mutant complemented with the entire fixNOQP operon looked very
similar to a wild type spectrum (Fig. 4; Bj4504). The mutants
showed either weak (Bj4528 fixQ), intermediate (Bj3618 fixP::aphII) or severe defects (Bj4503
fixNOQP,
Bj4526
fixN, Bj4518
fixO) in cytochrome b and c contents. The spectra of the
fixN and
fixO mutants were almost identical to those of
the operon deletion mutant, with a strong decrease and a slight red
shift of the cytochrome c peak and a partial decrease of b-type cytochromes. The cytochrome b-specific
shoulder was resolved in these mutants into two components, one at 558
nm and the other at 563 nm. The shape of the
fixQ mutant
spectrum was similar to that of the wild type; however, the cytochrome c peak appeared somewhat decreased. The fixP::aphII mutant exhibited a spectrum with almost normal amounts of
cytochrome b, but strongly decreased levels of cytochrome c. The results indicate that the B-type hemes of FixN were
present in the
fixQ and fixP::aphII mutants.
Figure 4:
Visible absorption difference spectra
(dithionite-reduced minus air-oxidized). Membrane proteins
(0.5 mg ml) solubilized with dodecyl maltoside from
the following B. japonicum strains were tested: Bj4504 (fixNOQP
by complementation), Bj4503
(
fixNOQP), Bj4526 (
fixN), Bj4518
(
fixO), Bj4528 (
fixQ), and Bj3618 (fixP::aphII).
In summary, the results from Western blots, heme stains and visible
light spectroscopy implied that (i) complementation of an operon
deletion with the wild type fixNOQP operon was successful;
(ii) FixN, FixO and FixP were absent in the fixN and
fixO mutants; (iii) the
fixQ mutation had
no effect on the presence of the FixN, FixO and FixP subunits except
perhaps a slight reduction in the amount of FixP (Fig. 2, lane 7, and the difference spectrum in Fig. 4); and
(iv) FixN and FixO, but not FixP, were present in the fixP::aphII mutant.
As shown
previously(2) , the cbb-type oxidase is
essential for symbiotic nitrogen fixation because it supports energy
conservation under the extremely microaerobic conditions that prevail
in soybean root nodules. Thus, we analyzed the ability of the
single-gene deletion mutants to fix nitrogen in symbiosis (Fix
phenotype; Table 3). Soybean seedlings were inoculated with the B. japonicum mutant strains, and nitrogenase activity was
measured as acetylene reduction activity 23 days after infection. While
the fixNOQP
strain and the
fixQ mutant were Fix
, the
fixN,
fixO and fixP::aphII mutants showed between 0
and 5% residual Fix activity. Nodules infected with the latter mutant
strains were greenish inside, indicating the absence of functional
leghemoglobin. Our results suggest that the FixN, FixO and FixP
proteins, but not the FixQ protein, are essential to energetically
support nitrogen fixation in root nodules.
To study the pathway for assembly of multisubunit membrane
complexes is inherently difficult. Lack of a single subunit often
causes complete absence of the entire complex because membrane proteins
may be rapidly degraded unless they find their correct partner
subunit(s) to become stabilized by protein-protein interaction. For
examples, the assembly of the FF
-ATPase of E. coli relies on the presence of both
and
subunits (35) , and cytochrome c
of the Rhodobacter capsulatus and P. denitrificans ubiquinol-cytochrome c oxidoreductase is required to
maintain the stability of the other two subunits (cytochrome b, Rieske iron sulfur protein)(27, 36) .
Here we report on studies to elucidate how assembly of the B.
japonicum cbb-type cytochrome oxidase takes place. The
approach was to knock out separately each of the four genes in the fixNOQP operon, and then we assessed the presence or absence
of the other gene products in the cytoplasmic membrane. It was
mandatory to construct in-frame deletions and ensure that genes located
downstream of the deleted gene were expressed at normal levels. In
fact, none of the deletions showed any polarity effects on the
downstream genes.
The mutations analyzed here affected cytochrome cbb formation to different extents, allowing us to
draw conclusions with respect to subunit composition, function and
assembly. Surprisingly, the
fixQ mutation did not cause
any severe deficiencies even though (i) a fixQ-like gene is
conserved in all organisms examined for the presence of fixNOQP-like operons (2, 3, 4, 5, 6) and (ii) fixQ was shown here to be transcribed and translated. Hence,
it is clear that the 54-amino acid FixQ protein is not essential for
the formation and function of the cbb
oxidase
complex. However, this does not exclude the possibility that FixQ is
bound to the oxidase, perhaps even in substoichiometric amounts. The
question of whether or not FixQ is part of the complex will be answered
only when substantial quantities of highly purified cytochrome cbb
have become available. The partially purified
oxidase preparation reported recently (11) was not of
sufficient quantity to identify a protein of the expected molecular
mass of FixQ, i.e. approximately 6 kDa. In this context, we
find it intriguing that the recent three-dimensional structure
determination of P. denitrificans cytochrome aa
has uncovered the presence of an unprecedented 60-amino acid
peptide whose functional role is also not known(37) .
The
FixN and FixO proteins, as opposed to FixQ, are absolutely essential
for the function and assembly of cytochrome cbb.
Neither FixN and FixO nor the FixP protein were detectable in membranes
isolated from
fixN and
fixO mutants. This
supports previous biochemical data obtained with purified cbb
oxidase from B. japonicum and two Rhodobacter species (8, 9, 11) which
showed that it is a three-subunit enzyme complex consisting of FixN-,
FixO-, and FixP-like proteins. Interestingly, in the B. japonicum
fixP insertion mutant we detected almost wild type levels of the
FixN and FixO proteins, whereas only FixP itself was absent. Moreover,
the mutant had wild type levels of cytochrome b, but a
decreased level of cytochrome c probably due to the absence of
FixP. Notably, FixO was shown by heme-staining after SDS-PAGE to be
present as mature holocytochrome c in the fixP mutant. The results are reminiscent of studies with the cytochrome bc
complex, in which cytochromes b and c
were still detectable in a mutant defective in
the gene for the Rieske iron-sulfur protein (36) .
Taken
together, our results led us to propose an ordered biogenesis pathway
for the cbb-type cytochrome oxidase. After
translation of the individual subunit polypeptides from the fixNOQP mRNA, FixN and FixO are probably the first to be inserted into the
membrane to form an apparently stable FixNO core complex. This primary
nucleation step occurs also in the absence of FixP and, therefore,
precedes assembly of the FixNO core with the FixP protein. The fact
that not even traces of FixN plus FixP and FixO plus FixP are
detectable in
fixO and
fixN mutants,
respectively, suggests that the subunits are degraded extremely rapidly
when assembly in the membrane is not possible for lack of cognate,
stabilizing protein partners.
In the aerobic respiratory chain of B. japonicum the bc complex appears to be
a nucleation center for the formation and stable maintenance of the
subsequent electron transport cytochromes CycM and aa
, which are thought to be organized in a bc
CycM
aa
supercomplex(24, 30, 32) . By contrast,
the FixNOP proteins do assemble also in the absence of, and therefore
independently from, the bc
complex.
The
phenotypical analyses of the mutants also help toward understanding the
organization of the microaerobic electron transport chain in B.
japonicum. It is now well established that under microaerobic
conditions electrons are transferred from ubiquinol to the bc complex that is also used in the aerobic
respiratory chain(30, 32) . The Fix
phenotype of a fixNOQP mutant has been interpreted to
mean that the oxidase encoded by these genes participates in the
microaerobic branch of respiratory electron transport(2) . This
is strongly supported by the high affinity for O
(K
= 7 nM) of this
oxidase(11) . Moreover, the structural predictions made for the
FixN protein imply that it corresponds to a heme-copper oxidase subunit
I, the component where oxygen is reduced. The two other components,
FixO and FixP, are likely to mediate electron transfer between the bc
complex and the FixN oxidase subunit. Midpoint
redox potentials have been determined for subunits of the homologous R. capsulatus cbb
-type oxidase. Values of 265 mV
and 320 mV were determined for the 32- and 28-kDa c-type
cytochromes, the ccoP and ccoO gene
products(8, 9) . CcoP, and by analogy FixP, is
therefore likely to mediate electron transfer between the bc
complex and CcoO (FixO). This would be a role
comparable to that of the membrane-bound CycM protein in the aerobic
respiratory branch of B. japonicum(32, 34) .
Since FixO is closely associated with FixN, it is the best candidate to
donate the electrons directly to FixN. In the case of the microaerobic
respiratory branch, the electrons may be transferred from the bc
complex to FixP, and from there to FixO and
FixN, where they are finally used for the reduction of molecular oxygen
to water.