From the Department of Microbiology and ** Centre for
Magnetic Resonance, The University of Queensland, Brisbane 4072, Australia and the ¶ Dipartimento di Biologia, Università di
Bologna, 40126 Bologna, Italy
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ABSTRACT |
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The dorC gene of the dimethyl
sulfoxide respiratory (dor) operon of Rhodobacter
capsulatus encodes a pentaheme c-type cytochrome that
is involved in electron transfer from ubiquinol to periplasmic dimethyl
sulfoxide reductase. DorC was expressed as a C-terminal fusion to an
8-amino acid FLAG epitope and was purified from detergent-solubilized membranes by ion exchange chromatography and immunoaffinity
chromatography. The DorC protein had a subunit
Mr = 46,000, and pyridine hemochrome analysis
indicated that it contained 5 mol heme c/mol DorC polypeptide, as
predicted from the derived amino acid sequence of the dorC gene. The reduced form of DorC exhibited visible absorption maxima at
551.5 nm ( The ability to use Me2SO and
trimethylamine-N-oxide
(TMAO)1 as electron acceptors
is widespread among facultative aerobic bacteria and the organization
of the Me2SO and TMAO respiratory chains is now well
defined (1). In the Me2SO respiratory system of purple
photosynthetic bacteria such as Rhodobacter capsulatus, electrons are transferred from primary dehydrogenases via the ubiquinone pool to a periplasmic Me2SO reductase (2).
Recent sequence and mutational analysis of the Me2SO
respiratory gene cluster from photosynthetic bacteria has identified a
pentaheme c-type cytochrome (DorC) as the likely mediator of
electron transfer from ubiquinol to Me2SO reductase
(3).2 The TMAO respiratory
(Tor) system of Escherichia coli (5) is very similar to the
Dor system of R. capsulatus, and they differ from the
Me2SO respiratory (Dms) system of E. coli. The
Me2SO reductase (DmsABC) of E. coli can be
purified as a menaquinol-oxidizing Me2SO reductase complex
that lacks c-type cytochromes (6). Me2SO
reductase from R. capsulatus has been purified as a
monomeric protein containing a pterin molybdenum cofactor (Moco) as its only prosthetic group (7). This property of Me2SO reductase has many advantages for spectroscopic characterization of Moco, but a
major deficiency has been the lack of a physiologically relevant
electron donor. To overcome this problem we describe in this paper the
purification and characterization of DorC.
The derived amino acid sequence of Rhodobacter DorC
indicated that it was related to members of the NirT class of tetraheme c-type cytochromes (3).2 However, DorC is
predicted to be a pentaheme cytochrome with a fifth c-type
heme binding motif in the C-terminal polypeptide, which is absent from
the tetraheme members of the NirT class. Almost all of the members of
the NirT class are involved in an anaerobic respiratory pathway, and
their role appears to be to catalyze electron transfer from the Q-pool
to a periplasmic terminal reductase (8). Although molecular genetic
studies have provided an insight into the function and likely
properties of cytochromes of the NirT class, very little is known about
their biochemical and spectroscopic properties.
Expression and Purification of DorC--
A 1.4-kilobase DNA
fragment containing the complete dorC nucleotide sequence
except for the stop codon and including the upstream regulatory
sequences of the dor operon was amplified by polymerase chain reaction using plasmid pALS3 as a template.2 The
forward primer was designed so that a BamHI site would be synthesized while the reverse primer directed the synthesis of an
EcoRI site plus a sequence to include a C-terminal 8-amino acid FLAG epitope (9). The 1.4-kilobase polymerase chain reaction product was cloned into pDSK519 (10) to create plasmid pALS300. pALS300
was transferred from E. coli S17-1 to R. capsulatus 37b4 by conjugation (11).
DorC was expressed in 2l R. capsulatus cells grown for
24 h under phototrophic conditions in RCV medium (12) in the
presence of 60 mM Me2SO. Membranes were
prepared from the cells using a French Press as in Ref. 2 and were
resuspended in Tris-buffered saline. Proteins were solubilized by
treatment of membranes with 4% Triton X-100 for 90 min at room
temperature. The mixture was then centrifuged (145,000 × g, 90 min, 4 °C), and the soluble fraction was collected.
The soluble fraction containing DorC (containing approximately 100 mg
of protein) was dialyzed against 50 mM Tris-HCl, pH 8.0, 0.2% Triton X-100 and then charged onto a DEAE-Trisacryl column (bed
volume, 15 ml), equilibrated with the same buffer. DorC was eluted
using a 100-ml linear gradient of NaCl from 0 to 1 M. The
DorC-enriched fraction from the anion exchange chromatography was
dialyzed against Tris-buffered saline plus 0.2% Triton X-100 and then
charged onto a 2-ml column containing FLAG affinity resin (Eastman
Kodak). Bound DorC was eluted with 0.1 M glycine, pH 3.5, 0.2% Triton X-100. 2-ml fractions were collected, and the pH was
immediately raised by addition of 50 µl 1 M Tris-HCl, pH 8.0.
Spectroscopy and Redox Potentiometry--
Optical spectra of
DorC were collected using a Hitachi U-3000 spectrophotometer. Pyridine
hemochrome analysis of DorC was carried out according to the method of
Berry and Trumpower (13). For reduction of DorC with duroquinol, the
cytochrome was incubated with 2 units ml Protein Methods--
SDS-PAGE analysis of proteins and Western
blotting using an anti-FLAG antibody were performed as in Refs. 15 and
16. Protein concentration was determined as in Ref. 17.
Expression and Purification of DorC--
A plasmid for the
expression of DorC with an 8-amino acid FLAG epitope attached to the C
terminus was constructed as described under "Experimental
Procedures." This plasmid was expressed in R. capsulatus
cells grown phototrophically in the presence of Me2SO.
Membrane and soluble fractions were prepared, and production of DorC
was monitored by Western blotting. A single immunoreactive polypeptide
with a molecular mass of 46 kDa was observed; this was the expected
size of the DorC-FLAG polypeptide (data not shown). The putative DorC
polypeptide was located exclusively in the membrane fraction. Several
detergents were tested for their ability to solubilize DorC. Triton
X-100 proved to be the most efficient, but even this detergent failed
to completely solubilize DorC because an immunoreactive polypeptide
could still be detected in the detergent-insoluble membrane fraction.
The detergent-solubilized fraction was applied to a DEAE-Trisacryl
anion exchange column. DorC was eluted from the column at about 450 mM NaCl. The fractions containing DorC were then applied to
a FLAG-affinity resin and eluted as described under "Experimental
Procedures." The pooled fractions containing the colored cytochrome
were analyzed by SDS-PAGE. Fig. 1 shows
the presence of a single polypeptide Mr = 46,000 corresponding to DorC. This protein cross-reacted with the anti-FLAG
antibody in Western blots and stained for heme-dependent
peroxidase activity (data not shown), confirming that the DorC-FLAG
polypeptide had been purified.
Optical Spectroscopy of DorC--
Fig.
2 shows a dithionite-reduced minus
ferricyanide-oxidized spectrum of DorC. Absorption peaks at 551.5 nm
(
The number of heme groups per DorC polypeptide was determined by
pyridine hemochrome analysis. Fig. 3
shows hemichrome and hemochrome spectra of DorC in alkaline pyridine.
The presence of an absorption maximum at 550 nm in the pyridine
hemochrome spectrum (Fig. 3) confirmed that DorC contained only
c-type hemes. Using the method of Berry and Trumpower (13),
it was calculated that there were 4.9 mol c-type heme/mol
DorC. This confirms that DorC is a pentaheme, as indicated from the
primary structure analysis.2
Determination of the Midpoint Redox Potential of the Heme Centers
of DorC--
The thermodynamic properties of the heme centers in DorC
were analyzed by redox potentiometry. It was observed that during reductive titration no reduction of the cytochrome was seen until the
ambient redox potential (at pH 7.0) was below 0 mV. This is consistent
with the observation that sodium ascorbate (Em = +65 mV)
did not reduce DorC as seen in reduced minus oxidized
difference spectra (data not shown). Fig.
4 shows the change in absorbance of the a
band (551.5 nm minus 540 nm as the reference wavelength) of DorC as a
function of the ambient redox potential. The data obtained could be
fitted to a five-component Nernstian curve (n = 1) with
midpoint potentials (Em7.0) of DorC-dependent Electron Transfer from Duroquinol to
Me2SO Reductase--
DorC is the only protein encoded by
the dor operon that could act as a mediator of electron
transfer between ubiquinol and Me2SO
reductase.2 Before reconstitution of this electron transfer
pathway was attempted, the ability of duroquinol to reduce DorC was
investigated. A duroquinol-reduced spectrum of DorC indicated that at least some of the heme centers of
the protein had been reduced (data not shown).
For the reconstitution of electron transfer from quinol to
Me2SO reductase DorC was titrated with an approximately
stoichiometric amount of duroquinol under anaerobic conditions. This
led to an increase in absorbance at 551.5 nm (Fig. 5). Upon addition of Me2SO reductase there was a decrease in the absorption of
the DorC from R. capsulatus is the first pentaheme
cytochrome in the NirT class to be purified and characterized. The
presence of photosynthetic pigments in the membrane of R. capsulatus made the detection of the native cytochrome very
difficult and necessitated the strategy of attaching a FLAG peptide as
a tag for detection and affinity purification. The protein was purified
from R. capsulatus because all of our attempts to express
DorC in E. coli were
unsuccessful.3 DorC was
located exclusively in the membrane of R. capsulatus, and
this would be consistent with a role in ubiquinol oxidation. In
vivo, it is expected that DorC would be a peripheral membrane protein anchored to the membrane by its hydrophobic N terminus, and
this explains why detergents were required to solubilize the protein.
The optical spectroscopy of DorC identified an absorption maximum at
551.5 nm for the There has been intense interest in the structure and mechanism of
Me2SO reductase (4, 24, 25), and many questions remain regarding the route of electron transfer to the molybdenum atom. It
seems almost certain that one of the molybdopterin guanine dinucleotide
moieties of Moco will act as a conduit for electrons from DorC. The
purification of DorC that we have described opens the way for
experiments to look at electron transfer to Me2SO reductase
using the physiological electron donor. This may help resolve some
current controversies relating to the mechanism of Me2SO
reductase by avoiding the need to use dithionite and viologens as
electron donors. Although we showed that DorC was reduced by duroquinol
and that reduced DorC was oxidized by Me2SO reductase, it
was not possible to use DorC to catalyze steady state electron transfer
between duroquinol and Me2SO reductase. The reason is that
Me2SO reductase itself exhibited
duroquinol-Me2SO oxidoreductase activity.3
However, under physiological conditions DorC would be required for the
oxidation of ubiquinol-10, which, unlike water-soluble duroquinol, is
buried within the hydrophobic domain of the cytoplasmic membrane and
thus is inaccessible to periplasmic Me2SO reductase.
-band), 522 nm (
-band), and 419 nm (Soret band). Redox
potentiometry of the heme centers of DorC identified five components
(n = 1) with midpoint potentials of
34,
128,
184,
185, and
276 mV. Despite the low redox potentials of the
heme centers, DorC was reduced by duroquinol and was oxidized by
dimethyl sulfoxide reductase.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1 Clostridial
diaphorase, 25 µM duroquinone and 200 µM
NADH. Redox potentiometry of DorC was performed by the method of Dutton
(14). Absorption spectra were recorded between 500 and 600 nm using a
Jasco 7850 UV-visible spectrophotometer. Redox titrations were performed with dithionite as reductant and ferricyanide as oxidant in
the presence of the following mediators: 2.5 µM
benzoquinone (Eo' = +280 mV), 2.5 µM 1,2-naphthoquinone
(Eo' = +145 mV), 2.5 µM 1,4-naphthoquinone (Eo' = +60
mV), 20 µM duroquinone (Eo' = +5 mV), 20 µM
1,4-dihydroxynaphthoquinone (Eo' =
145 mV), 5 µM anthroquinone-2-sulfonate (Eo' =
225 mV), and 5 µM
benzylviologen (Eo' =
350 mV). The absorbance change at 551.5-540 nm
was plotted against redox potential, and theoretical Nernstian curves
were fitted to the data using Sigma Plot.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Coomassie-stained SDS-PAGE gel of purified
DorC. Lane 1, molecular mass markers; lane 2, 8 µg of purified DorC.
-band), 522 nm (
-band), and 419 nm (Soret band) were typical of
a reduced cytochrome. The absorption minimum at 408 nm represents the
Soret absorption band of the oxidized form of DorC. A spectrum of the resting form of DorC exhibited this 408 nm peak, and the absence of the
-band and
-band of the reduced cytochrome indicates that the
DorC, as prepared, is in a fully oxidized state.
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Fig. 2.
Dithionite reduced minus ferricyanide
oxidized difference spectrum of DorC.
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Fig. 3.
Pyridine hemochrome (A) and
pyridine hemichrome (B) spectra of DorC.
34 ± 2,
128 ± 2,
184 ± 3,
185 ± 3, and
276 ± 3 mV.
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Fig. 4.
Redox potentiometry of DorC (12 µM). Symbols represent the results
of two separate titrations. The solid line is a theoretical
curve generated using the Nernst equation for five n = 1 oxidation-reduction processes with midpoint redox potentials 34,
128,
184,
185, and
276 mV with relative contributions to the
total absorbance signal of 10, 22, 15, 30, and 23%,
respectively.
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Fig. 5.
A, increase in the absorbance of the
-band of DorC with time (0-14 min) upon incubation of 8.0 µ
M DorC in 20 m M HEPES-NaOH, pH 7.2, 1 m
M EDTA, 0.1% Triton X-100 with 2 units ml
1
diaphorase, 20 µM NADH, and 10 µM
duroquinone. At time 15 min, 3 µM Me2SO
reductase was added to the reduced DorC, and the decrease in the
absorbance of the
-band was measured from time 16 min. B,
increase in absorbance at 551.5 nm against time. C, decrease
in absorbance at 551.5 nm against time.
-band of DorC (Fig. 5), indicating that electron transfer from DorC to Me2SO reductase had occurred. Control experiments
showed that the oxidation of DorC was specifically dependent upon
Me2SO reductase.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-band. This is a symmetrical peak with very little
complexity, and it suggests that although the midpoint redox potentials
of the heme centers differ, it is likely that each possesses the same
fifth and sixth ligands. There was no evidence for high spin five
coordinate heme with an optical band beyond 600 nm as seen in
cytochrome c' for example (18). Redox potentiometry was
consistent with the presence five heme centers, all of which have
midpoint redox potentials below 0 mV. These are surprisingly low values
for a protein that is thought to act as an electron acceptor from
ubiquinol because the Eo' for the ubiquinol/ubiquinone redox couple is
+40 mV (19). However, it was observed that water-soluble duroquinol
could reduce DorC. The thermodynamic quantity described in this paper
is the "macroscopic" potential of the heme centers of DorC (20,
21). These parameters are not influenced by electron/electron
cooperativity between heme centers. However, it has been shown that in
the tetraheme cytochrome c3 from
Desulfovibrio vulgaris each heme redox potential is
dependent on the oxidation state of the other three hemes (22), and
this can affect the thermodynamic properties of a multi-heme protein
(21). This can lead to a variety of potential microscopic heme-heme
interacting potentials that may be higher than the macroscopic potentials. The ability of DorC to be reduced by duroquinol can be
rationalized if the heme with the highest potential (
34 mV) is
reducible by duroquinol. Under physiological conditions this would
suggest that the DorC protein would be reduced when the Eh
of the ubiquinol/ubiquinone pool was lower than the Eo' for the
ubiquinol/ubiquinone redox couple. Such a situation could occur under
phototrophic (anaerobic) conditions in the presence of reduced carbon
sources (23). Further work to demonstrate and analyze heme-heme
interactions in DorC using EPR and/or NMR spectroscopy is now required.
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ACKNOWLEDGEMENTS |
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We thank Bruno Guigliarelli and David Richardson for helpful comments and discussion.
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FOOTNOTES |
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* This work was supported by a grant from the Australian Research Council (to A. G. M. and G. R. H.) and an Australian Research Council International project grant (to A. G. M.).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.
§ Recipient of an Australian Postgraduate Award.
Supported by Ministero Università e Ricerca Scientifica
Technologica of Italy.
To whom correspondence should be addressed. Fax:
61-7-3365-4620; E-mail: mcewan{at}biosci.uq.edu.au.
2 Shaw, A. L., Knaeblein, J., Leimkuhler, S., Hanson, G. R., Klipp, W., and McEwan, A. G. (1999) Microbiology, in press.
3 A. L. Shaw and A. G. McEwan, unpublished observations.
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ABBREVIATIONS |
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The abbreviations used are: TMAO, trimethylamine-N-oxide; PAGE, polyacrylamide gel electrophoresis.
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