Hooper2 1 Department of Biology, Black Hills State University, Spearfish, SD, USA; 2 Department of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota, MN, USA The he
Trang 1Cytochrome P460 of Nitrosomonas europaea
Formation of the heme-lysine cross-link in a heterologous host and mutagenic
conversion to a non-cross-linked cytochrome c ¢
David J Bergmann1and Alan B Hooper2
1 Department of Biology, Black Hills State University, Spearfish, SD, USA; 2 Department of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota, MN, USA
The heme of cytochrome P460 of Nitrosomonas europaea,
which is covalently crosslinked to two cysteines of the
polypeptide as with all c-type cytochromes, has an additional
novel covalent crosslink to lysine 70 of the polypeptide
[Arciero, D.M & Hooper, A.B (1997) FEBS Lett 410, 457–
460] The protein can catalyze the oxidation of
hydroxyl-amine The gene for this protein, cyp, was expressed in
Pseudomonas aeruginosastrain PAO lacI, resulting in
for-mation of a holo-cytochrome P460 which closely resembled
native cytochrome P460 purified from N europaea in its
UV-visible spectroscopic, ligand binding and catalytic
properties Mutant versions of cytochrome P460 of
N europaeain which Lys70 70 was replaced by Arg, Ala, or
Tyr, retained ligand-binding ability but lost catalytic ability
and differed in optical spectra which, instead, closely
resembled those of cytochromes c¢ Tryptic fragments
con-taining the c-heme joined only by two thioether linkages were observed by MALDI-TOF for the mutant chromes P460 K70R and K70A but not in wild-type cyto-chrome P460, consistent with the structural modification of the c-heme only in the wild-type cytochrome The present observations support the hypothesized evolutionary relationship between cytochromes P460 and cytochromes c¢
in N europaea and M capsulatus [Bergmann, D.J., Zahn, J.A., & DiSpirito, A.A (2000) Arch Microbiol 173, 29–34], confirm the importance of a heme-crosslink to the spectro-scopic properties and catalysis and suggest that the crosslink might form auto-catalytically
Keywords: cytochrome c¢; cytochrome P460; hydroxyl-amine; nitric oxide, Nitrosomonas
Cytochromes P460 are mono-heme cytochromes
character-ized by Soret absorption maxima at approximately 435, 460
and 450 nm in the ferric, ferrous and ferrous-CO forms,
respectively [1–4] Although the protein catalyzes the
oxidation of hydroxylamine, its physiological role has not
been clearly elucidated
Cytochrome P460 of Nitrosomonas is a homo-trimer [2,3]
or possibly -dimer [1] of 18 kDa subunits Several unique
features of the optical spectra of cytochrome P460 are
shared by heme-P460, the active site heme of
hydroxyl-amine oxidoreductase (HAO) of N europaea HAO is a
homo-trimer of octa-heme subunits which catalyzes high
rates of dehydrogenation of hydroxylamine [5–8] In addition
to two thioether linkages to cysteine residues, active site
hemes of cytochrome P460 or HAO have a third covalent
linkage from a heme to Tyr467 in the adjacent subunit of
HAO [7,9] or from the heme P460 to Lys70 in cytochrome
P460 [10], respectively However, the two enzymes have no
similarity in amino acid sequence [11,12] Cytochromes P460
have been characterized from the autotrophic ammonia oxidizing bacterium Nitrosomonas europaea of the b-sub-division proteobacteria [1] and from the methane oxidizing bacterium Methylococcus capsulatus Bath of the c-subdivi-sion [14] The amino acid sequences of cytochromes P460 from both N europaea and M capsulatus Bath have signi-ficant similarity to that of cytochrome c¢ from M capsulatus Bath, suggesting a possible evolutionary link between the three cytochromes [13] Cytochromes c¢, which are found in a wide variety of photosynthetic, denitrifying, and methano-trophic bacteria, are homo-dimers of 16 kDa to 18 kDa subunits which have one penta-coordinate c-type heme capable of binding small ligands such as NO or CO [14,15] Although cytochrome c¢ of M capsulatus has spectroscopic properties that are unique to the other cytochromes c¢ it exhibits only weak homology in primary structure with the majority of cytochromes c¢ [13]
We hypothesized that if the ancestral form of cytochrome P460 was a cytochrome c¢ which evolved by the acquisition
of a third covalent crosslink to the heme (causing it to gain the ability to catalyze hydroxylamine oxidation), then mutants of cytochrome P460, in which the Lys70 is replaced
by an amino acid residue unable to crosslink to the active site heme, might well have spectroscopic, ligand-binding and catalytic properties similar to cytochromes c¢ In this paper, we report the expression in Pseudomonas aeruginosa PAO lacI of wild-type cytochrome P460 and site-directed mutants in which Lys70 was replaced by arginine, alanine or tyrosine The wild-type cytochrome P460 expressed in
Correspondence to A B Hooper, D epartment of Biochemistry,
Molecular Biology and Biophysics, University of Minnesota,
St Paul, MN 55108, USA.
Fax: + 1 612 625 5780, Tel.: + 1 612 624 4930,
E-mail: hooper@cbs.umn.edu
Abbreviations: HAO, hydroxylamine oxidoreductase.
(Received 27 December 2002, revised 19 February 2003,
accepted 3 March 2003)
Trang 2P aeruginosa had properties very similar to that from
N euroapea.However, the mutant cytochromes P460 had
properties very different from wild-type cytochrome P460
and similar to cytochromes c¢
Materials and methods
DNA techniques
The gene encoding cytochrome P460 of N europaea, cyp,
was amplified by PCR from a 7.8-kb BamHI fragment of
genomic DNA which had previously been subcloned into
the plasmid vector pUC119 [16] The forward primer,
5¢-GCTACCATATGAAAACAGCTTGGTAGGT-3¢,
en-compassed the ATG start codon of cyp and contained a 5¢
extension with an NdeI site The reverse PCR primer,
5¢-CCTGATTCGTTCTGCTACCT-3¢, bound to a region
just downstream of a cyp and a native SmaI–XmaI site
PCR reaction mixture (100 lL) was used (PCR Super Mix,
Life Technologies, Inc., Gaithersburg, MD, USA)
contain-ing 0.2 nmol of each primer, 2 ng template, 0.2 mMdNTPs,
50 mM Tris/HCl (pH 8.4), 1.5 mMMgCl, and 1.0 U Taq
DNA polymerase After denaturation of the PCR mixture
at 94C for 5 min, 30 cycles of 94 C for 30 s, 60 C for
30 s, and 72C for 30 s were performed, and the reaction
incubated for 7 min at 72C and stored at 4 C
The PCR product was purified using a spin-column kit
(Qiagen, Inc., Valencia, CA, USA) The PCR product and
the expression vector pUCPNde [17] were digested with
NdeI and XmaI as directed by the manufacturer (Promega,
Inc., Madison, WI, USA) The digested PCR product and
expression vector were ligated and transformed into frozen
competant Escherichia coli strain DH5aF¢IQ cells (Life
Technologies) using standard methods [18] Transformed
colonies were grown on LB (Luria–Bertani) media with
100 lgÆmL)1ampicillin, and plasmid DNA harvested by the
alkaline lysis technique [18] The orientation of cyp
subcloned in the expression vector was confirmed by
dideoxy dye-primer cycle sequencing using an ABI Model
377 DNA Sequencer at the University of Minnesota AGAC
sequencing facility The resulting plasmid, pUCYP2,
con-tained the wild-type cyp gene downstream of the lac
promoter and ribosome-binding site of the vector
Three site directed mutants of cyp, in which the codon,
AAA, encoding Lys70 of cytochrome P460 is converted to
AGA (Arg), GCA (Ala), or TAT (Tyr) were produced from
pUCYP2 with the Transformer site directed mutagenesis kit
(Clontech, Inc., Palo Alto, CA, USA) using the method of
Deng and Nicloff [19], as directed by the manufacturer The
selection oligonucleotide, 5¢-AAATGCTTCAATGATAT
CGAAAAAGGAAG-3¢, converted a unique SspI site on
the vector to an EcoRV site The mutagenesis
oligonucleo-tides were 5¢-GTAACTGTAAGAGAACTGGTCAC-3¢
(Lys70 to Arg), 5¢-GTAACTGTAGCAGAACTGGTCA
G-3¢ (Lys70 to Ala), and 5¢-GGTAACTGTATATGAA
CTGGTCAG-3¢ (Lys70 to Tyr) The resulting plasmids,
pUCYPKR, pUCYPKA, and pUCYPKY, were
trans-formed into cells [20] The plasmids pUCYP2, pUCYPKR,
pUCYPKA, and pUCYPKY were each introduced into
Pseudomonas aeruginosastrain PAO-LacI by
electropora-tion using an Electroporator 2510 (Eppendorf Co.,
West-bury, NY, USA) as described by Cronin and McIntire [17]
Growth of cells and purification of cytochrome P460 Cells of P aeruginosa PAO lacI-containing plasmids pUCYP2, pUCYPKR, pUCYPKA, or pUCYPKY were grown and periplasmic extracts prepared as described by Cronin and McIntire [17] The periplasmic extract was dialyzed in Union Carbide 5 cm-wide dialysis bags (m cut-off < 25 kDa) overnight at 5C against 6 L of KPibuffer (50 mMpotassium phosphate, pH 7.5) Ammonium sulfate was then added to the periplasmic extract to 75% saturation and stirred for 45 min at 5C before centrifugation at
15 000 g for 15 min at 5C The pellet was discarded, and the supernatant brought to 100% saturation in ammonium sulfate and stirred for 45 min at 5C After centrifugation at
15 000 g for 15 min at 5C, the pellet was resuspended in
20 mL KPibuffer and dialyzed overnight against 4 L of KPi buffer KCl was then added to 200 mM and the sample concentrated to 1 mL on an Amicon stirred filtration cell with a YM 10 membrane and Centricon 10 microconcen-tration devices (Amicon-Grace Co., Danvers, MA, USA) The sample was added to a 2.5-cm diameter· 110 cm long column of Sephadex G-100 (Sigma Chemical Co., St Louis,
MO, USA) equilibrated with 200 mM KCl in KPibuffer Cytochrome P460 eluted as a greenish-yellow band, free of endogenous c-type cytochromes from P aeruginosa, but still containing other, nonheme, protein contaminants (Fig 1)
Fig 1 SDS/PAGE (15% acrylamide/bisacrylamide) of wild-type cytochrome P460 from N europaea and P aeruginosa and mutant cytochromes P460 expressed in P aeruginosa (A) Stained with Coo-massie Blue R-250 (B) Stained for heme by the method of Goodhew
et al [30] Lanes 1 and 7, high range prestained molecular mass markers (Life Technologies); lane 2, wild-type cytochrome P460 purified from N europaea; lanes 3–6, cytochromes P460 expressed in and partially purified from the periplasm of P aeruginosa; lane 3, wild-type cytochrome P460; lane 4, cytochrome P460 K70R; lane 5, cytochrome P460 K70A; lane 6, cytochrome P460 K70Y.
Trang 3and had a Soret : 280 nm absorbance ratio of
approxi-mately 2.0
Optical absorption spectroscopy was performed with a
Hewlett-Packard 8452 diode-array spectrophotometer
(Agilent Technologies) Cytochrome P460 from N
euro-paea was prepared by D M Arciero, University of
Minnesota, St Paul, MN, USA, as described previously
[21] Cytochrome c552 was obtained from N europaea by
D M Arciero as described earlier [22] for use as an electron
acceptor in assays of hydroxylamine and hydrazine
oxida-tion by cytochrome P460 In these assays, the absorbance of
9 lMcytochrome c552 in 1 mL of KPibuffer at pH 7.5 was
monitored at 552 nm in the presence of substrate and
cytochrome P460 at 22C
SDS/PAGE, tryptic digests of cytochrome P460
and mass spectrometry
Approximately 0.5–1.0 nmole of cytochrome P460 was
denatured in 1· loading buffer [60 mMTris/HCl (pH 6.8),
10% v/v glycerol, 1% w/v SDS] for 30 min at room
temperature and was loaded onto a (15 : 0.4%)
acrylamide-bisacrylamide gel for electrophoresis at room temperature
using the Laemmli buffer system [23] The yellow or orange
cytochrome P460 band was excised from the gel and the
cytochrome digested within the gel slice with porcine
sequencing grade trypsin (Promega Corp., Madison WI,
USA) and then eluted from the slice as described by
Shevchenko et al [24] Prior to MALD I-TOF analysis, the
sample was desalted using C18 ZipTips using the protocol
described by the manufacturer (Millipore Corp., Bedford,
MA, USA), with the following modification: the elution
buffer was 75 : 25, acetonitrile/water, 0.1% trifluoroacetic
acid The instrument used for the collection of
Matrix-Assisted Laser Desorption/Ionization Time-of-Flight
(MALDI-TOF) mass spectrometric data was a Bruker
Biflex III, equipped with an N2-laser (337 nm, 3
nanosec-ond pulse length) and a microchannel plate detector The
data was collected in the reflectron mode, positive polarity,
with an accelerating potential of 19 kV Each spectrum is
the accumulation of 200 laser shots External calibration
was performed using human angiotensin II (monoisotopic
mass [MH+] 1046.5) and adrenocorticotropin hormone
(ACTH) fragment 18–39 (monoisotopic mass [MH+]
2465.2; Sigma Chemical Co., St Louis, MO, USA) The
matrix used for samples and standards was
cyano-4-hydroxycinnamic acid (4-HCCA; Hewlett-Packard, sold in
solution, in methanol) diluted 1 : 1 with 50 : 50,
acetonit-rile/nanopure water, 0.1% trifluoroacetic acid HPLC grade
acetonitrile was purchased from Fisher Scientific and
99+% spectrophotometric grade trifluoroacetic acid was
purchased from Aldrich, (Milwaukee, WI, USA) The
presence of heme in fragments was confirmed by the pattern
from the 54/56Fe isotope-distribution: a small secondary
peak was seen, which had a mass two units below the peak
in consideration The secondary peak was present only in
peaks reported in Table 2 to contain heme
Results
Expression of wild-type and mutant cytochrome P460 in
P aeruginosaPAO lacI, containing the plasmid pUCYP2
(which encodes the gene for wild-type cytochrome P460 from N europaea) expressed a cytochrome whose subunit molecular size, UV/visible spectral properties and reactivity with ligands and substrates were identical with those of cytochrome P460 purified from N europaea The yield of the cytochrome was low, however, ranging from 0.2 to 0.4 mg of cytochrome P460 per litre of cell culture based on the intensity of the Soret peak [3]
The migration pattern on SDS/PAGE gels for samples of cytochromes P460 are shown in Fig 1 All samples exhibited a heme-containing band of the same mobility as the subunit of purified wild-type cytochrome P460 produced
in Nitrosomonas The wild-type and K70R, K70A, and K70 mutant cytochromes P460 expressed in P aeruginosa eluted similarly on a Sephadex G100 column; they eluted later and well-separated from the P aeruginosa cyt c551 (Mr12 000) thus are likely to have the same quaternary structure as the wild-type expressed in N europaea
Optical spectroscopy Wild-type cytochrome P460 expressed in Pseudomonas was indistinguishable from wild-type cytochrome P460 expressed in Nitrosomonas [1,3] with respect to the following spectroscopic, ligand binding and catalytic properties The optical spectrum of ferric wild-type cytochrome P460 expressed in P aeruginosa (Fig 2A, Table 1) had a broad Soret peak at 434 nm, and shoulders at510 and 540 nm The dithionite-reduced cytochrome had Soret absorbance at
462 nm, the 510/540 nm shoulder was lost and small peaks
at 660 and 688 nm appeared When CO was bubbled though the dithionite-reduced cytochrome P460, the Soret peak shifted to 448 nm and the 660/688 nm peaks were lost The latter may have been replaced by weak bands at 620 and 670 nm Optical changes associated with the reduction
of cytochrome P460 by dithionite occurred in two phases, which are not understood mechanistically but are com-monly observed with cytochrome P460 from Nitrosomonas
A rapid reduction in absorbance at 434 nm and 500 nm and increase at 688 nm preceded a slow increase at 462 nm, which was completed in 20 min A few preparations of cytochrome P460 from P aeruginosa lacked the broad shoulder at 510–540 nm in the oxidized state This has also been observed with some preparations of cytochrome P460 from N europaea and can occur during storage (D M Arciero, unpublished observation)
The addition of several grains of potassium cyanide to ferric-cytochrome P460 expressed in P aeruginosa caused the Soret band to decrease slightly and shift to 442 nm (data not shown) Incubation of ferric-cytochrome P460 with
100 lM hydroxylamine in 50 mM phosphate solution,
pH 7.5, caused the Soret band to increase slightly while absorbance at 500 nm decreased and absorbance at approximately 620 nm increased (data not shown) Incuba-tion of ferric-cytochrome P460 with 100 lM hydrazine caused the Soret absorbance peak to initially shift to 438 nm and increase in intensity then greatly diminish over several minutes, while absorbance at 500 nm decreased and absorbance at 620 increased slightly (data not shown) Cytochrome P460 expressed in P aeruginosa catalyzed the oxidation of either hydroxylamine or hydrazine, using cytochrome c552 from N europaea as an electron acceptor
Trang 4In the presence of 5 mM hydroxylamine or hydrazine, respectively, turnover numbers as high as 10.8 or 0.3 mol cytochrome c552 reduced per minute per mole of cyto-chrome P460 were obtained These values are comparable
to values measured with cytochrome P460 from Nitroso-monas[3]
In mutant versions of cytochrome P460 expressed in
P aeruginosa, Lys70 was replaced by arginine, alanine, or tyrosine to form cytochromes P460 K70R, P460 K70A, or P460 K70Y, respectively None of the mutant cytochromes P460 catalyzed the oxidation of hydroxylamine using
N europaeacytochrome c552 as an electron acceptor nor did the optical spectra of any of the three mutant ferric-cytochromes change in the presence of hydroxylamine, hydrazine, or potassium cyanide The UV-visible spectra of the P460 mutants K70R, K70A, and K70Y were similar to each other but were strikingly different from those of wild-type cytochrome P460 produced in N europaea or
P aeruginosa (Fig 2B–D, Table 2) Mutant ferric-cyto-chromes P460 had Soret peaks in the range 392–404 nm, smaller broad peaks at 498 and 540 nm and an even smaller peak in the range 622–638 nm Mutant cytochromes P460 were rapidly reduced by dithionite and the spectra of the ferrous forms displayed Soret peaks in the range 432–
434 nm and small peaks at 552–554 nm and 590 nm In common with cytochromes c¢ they lacked distinct a and b peaks in the ferrous form After bubbling CO into solutions
of the dithionite-reduced mutant cytochromes P460, the Soret peak shifted to 416–418 nm and smaller peaks shifted
to 532–534 nm and 562–564 nm These spectral features are strikingly similar to those observed in cytochromes c¢ from Methylococcus capsulatus[25] and Paracoccus denitrificans [26]
Proteolysis and MALDI-TOF MS Tryptic fragments of cytochromes P460 were prepared and analyzed by MALDI-TOF mass spectrometry (Table 2) MALDI-TOF spectra of tryptic fragments of wild-type cytochrome P460 produced by either N europaea or
P aeruginosa(Table 2) were nearly identical This sugges-ted, again, that the wild-type cytochrome P460 is expressed
in its native crosslinked form in the heterologous host Heme-containing tryptic peptide fragments representing the cysteine-containing residues #130–145, NLPTAECA ACHKENAK (Mr2314.5), or residues #130–141, NLPTA ECAACHK (Mr1872.4) were not observed in the MALDI-TOF spectrum in digests of either of the wild-type
Table 1 Absorption maxima of cytochromes P460 produced in P aeruginosa Cytochromes P460 include K70 (wild-type), K70R, K70A and K70Y Data are from Fig 2.
Type of cytochrome P460
Absorption maxima (nm) Ferric cytochrome Ferrous cytochrome Ferrous + CO cytochrome K70 (wt) 434, 510, 540 462, 660, 688 448 (620, 670?)
K70R 392, 498, 540, 638 434, 554, 590 418, 534, 566
K70A 402, 498, 540, 622 432, 552, 590 416 532, 562
K70Y 404, 498, 540, 628 432, 554, 590 416, 534, 564
Fig 2 UV/visible spectra of cytochromes P460 expressed in wild-type
P aeruginosa The purified cytochromes are in 50 m M potassium
phosphate solution, pH 7.5 Spectra for resting state of cytochrome,
as isolated, (solid line); dithionite-reduced (dotted-and-dashed line);
dithionite-reduced cytochrome + CO (dotted line) (A) Wild-type
cytochrome P460 K70, (B) cytochrome P460 K70R, (C) cytochrome
P460 K70A, (D) cytochrome P460 K70Y Wavelength of peaks are
labeled for ferric-, ferrous- and ferrous + CO-cytochrome P460 in D,
C and B, respectively.
Trang 5cytochromes In addition, no detectable tryptic fragment
from a wild-type cytochrome P460 contained either of these
two heme-containing polypeptides cross-linked to another
peptide containing Lys70 It is not known why these tryptic/
MALDI-TOF fragments, which are predicted from the
structure of wild-type cytochrome P460, are absent The
chromophoric lysyl-heme-di-cysteinyl cross-linked
dipep-tide is known to be very labile and to require extreme care
for its isolation [10] Hence it is likely to have been degraded
to a family of fragments at some step in the analysis
Alternatively it may have been lost during elution from the
gel or desalting of the eluate or was not desorbed/ionized
during MS analysis Free heme was not observed in the
MALDI-TOF spectrum of tryptic fragments of the
wild-type cytochromes The appearance of some of the
Lys70-containing peptide of residues #64–78 (lacking heme) in
each of the spectra of the wild-type proteins might suggest
that the K70 crosslinks had not ever formed in some
molecules of cytochrome P460 or were broken during the
analysis
The tryptic peptides of two mutant cytochromes P460,
P460 K70R and P460 K70A, had nearly identical
MALDI-TOF spectra, however, their spectra were substantially
different from corresponding MALDI-TOF spectra of
wild-type cytochromes P460 (Table 2) The spectra of tryptic
peptides of mutant cytochromes P460 K70R and P460 K70A
contained a 617.2-Da fragment representing free heme and
fragments of Mr2314.5 (residues #130–145, NLPTAECA
ACHKENAK) or Mr1872.4 (residues #130–141, NLPTA
ECAACHK) representing heme bound to tryptic peptides
through thioether linkages to cysteine residues The presence
of the latter two fragments from the mutant cytochromes
P460 in contrast to the absence of heme-cysteinyl-linked
peptides in the fragments from the wild-type protein is in
keeping with an apparent heme lability imparted by the
crosslink to lysine in the wild-type protein [10] Fragments
representing the heme crosslinked to two peptides through thioether linkages to two cysteines and also to a third residue (such as Lys70) were not found by MALDI-TOF of tryptic peptides of P460 K70R or P460 K70A
Discussion
The product of expression of the gene encoding wild-type cytochrome P460 from N europaea in P aeruginosa was a cytochrome with catalytic and ligand-binding capabilities and UV-visible spectroscopic properties identical to cyto-chrome p460 expressed in N europaea This implies that formation of the covalent crosslink between Lys70 and the heme of cytochrome P460 is facilitated by an enzyme present in both Nitrosomonas and the heterologous host or
is, in fact, auto-catalytic
The presence, in addition to the lysyl-heme crosslink, of the two thioether bonds from heme vinyl groups to peptide cysteines means that cytochrome P460 can be thought of as
a modified c-cytochrome By comparison with typical c-type cytochromes, the spectra of ferric cytochrome P460 has a greatly red-shifted Soret maximum, low and broad 510 and
540 nm shoulders and a small maximum at 688 nm On reduction, the Soret shifts to 460 nm and the 500 and
688 nm peaks disappear but a and b maxima typical of c-type cytochromes do not appear The addition of CO causes the Soret maximum to shift to 450 nm The UV/ visible spectra of the ferric, ferrous or ferrous-CO forms of the mutant cytochromes P460, K70R, K70A, and K70Y, all lack the characteristic red-shifted 435, 460 or 450 nm absorption maxima of the wild-type cytochrome P460 thus confirming the role of the crosslink in determining the Soret spectrum Although catalytic activity was lost in the mutants, the ligand-binding capability and thus the penta-coordinate nature of the heme was conserved Significantly, typical c-cytochrome a and b maxima do not appear in the
Table 2 Relative intensities, as detected by MALDI, of identifiable tryptic fragments of cytochromes P460 from N europaea (NE) and P aeruginosa (PA) Cytochrome P460 70K (wild-type) isolated from NE or heterologously expressed in and purified from PA and mutants K70R and K70A heterologously expressed in and purified from PA Data were obtained as described in Materials and methods and analyzed by the methods of Wilkins et al [29] Residue number is based on the sequence of the mature protein.
Fragment M r P460–70K (NE) P460–70K (PA) P460-K70R (PA) P460-K70A (PA) Cytochrome P460 residues 2831.4 0.023 0.0 0.025 0.020 20–44
2314.5 0.0 0.0 0.193 0.125 130–145 + Heme 2247.6 0.105 0.104 0.093 0.079 79–100
2119.5 0.030 0.052 0.016 0.016 80–100
2016.6 0.059 0.058 0.017 0.019 20–37
1872.4 0.0 0.0 0.172 0.154 130–141 + Heme
1700.5 0.130 0.067 0.031 0.019 1–16
1632.5 0.183 0.133 0.031 0.032 unknown
1616.5 1.000 1.000 1.000 1.000 146–158
1594.3 0.413 0.362 0.073 0.065 45–57
833.4 0.433 0.683 0.072 0.068 38–44
Trang 6mutant proteins; rather, the ferric, ferrous and ferrous-CO
absorption spectra strongly resemble those of
cyto-chromes c¢ [25,26] It appears that, in the absence of the
lysine crosslink, the resulting heme environment of mutant
cytochrome P460 is typical of cytochromes c¢ rather than
the more common c-cytochromes A degree of sequence
similarity between the cytochromes P460 and c¢ has lead to
the hypothesis that cytochromes P460 may have evolved
from cytochromes c¢ [13] We note that the ancestor of
cytochromes c¢ and P460 could also have been a
catalyti-cally active and heme-crosslinked cytochrome P460-like
protein The present observations further document an
evolutionary relationship between the two proteins
In HAO the catalytic heme is crosslinked to a tyrosine of
the adjacent subunit [7] and forms the catalytic heme P460
with a ferrous absorption maximum of 460 nm As shown
here by their elution behavior in Sephadex G-100, the
wild-type or mutant cytochromes P460 appear also to be
oligomeric Thus intersubunit crosslinking would have been
at least theoretically possible The K to Y mutant of
cytochrome P460 was constructed for the present report to
test the remote possibility that a heme P460 derivative
homologous to heme P460 of HAO would be formed That
the chromophore was not formed may result from
differ-ences in the chemical nature of the lysyl- or tyrosyl- linkage
or the relative orientation of K70 and Y70 in the
cytochromes P460
In nature, the lysyl- or tyrosyl- to heme crosslink appears
to be of key importance to catalysis of electron and/or
proton removal from substrate [27] This is illustrated by the
difference between the dimeric nitrite reductase, NrfA, the
monomer of which is a penta-heme c-cytochrome lacking a
covalent crosslink to the catalytic heme [28] and
hydroxyl-amine oxidoreductase (a dehydrogenase), in which the
monomer is a octa-heme c-cytochrome possessing a tyrosine
crosslink to the catalytic heme Cytochromes c¢, lacking the
lysyl-heme crosslink, can reversibly bind but not transform
NO whereas cytochrome P460, having the lysine crosslink,
can bind and catalyze the transformation of NH2OH The
relevance of the lysyl-heme crosslink to catalysis is
suppor-ted here by the lack of catalytic ability in mutant
cytochromes P460 lacking the lysine crosslink
Acknowledgements
We thank Bradley Elmore, Ciarran Cronin, David Arciero, Mark
Whittaker, Michelle Wagner, and Leann Higgins for their help with this
project This research was funded by the National Science Foundation
(MCB-9723608).
References
1 Erickson, R.H & Hooper, A.B (1970) Preliminary
characteriza-tion of a variant CO-binding heme protein from Nitrosomonas.
Biochim Biophys Acta 275, 231–244.
2 Miller, D.J., Wood, P.M & Nichols, D.J.D (1984) Further
characterization of cytochrome P-460 in Nitrosonomas europaea.
J Gen Microbiol 130, 3049–3054.
3 Numata, M., Saito, T., Yamakazi, T., Fukumori, Y &
Yama-naka, T (1990) Cytochrome p460 of Nitrosomonas europaea:
further purification and further characterization J Biochem 108,
1016–1021.
4 Zahn, J.A., Duncan, C & DiSpirito, A.A (1994) Oxidation of hydroxylamine by cytochrome P-460 of the obligate methano-troph, Methylococcus capsulatus Bath J Bacteriol 176, 5879– 5887.
5 Hooper, A & Nason, A (1965) Characterization of hydro-xylamine-cytochrome c reductase from the chemoautotrophs Nitrosomonas europaea and Nitrosocystis oceanus J Biol Chem.
240, 4044–4057.
6 Arciero, D.M & Hooper, A.B (1993) Hydroxylamine oxido-reductase is a multimer of an octaheme subunit J Biol Chem.
268, 14645–14654.
7 Igarashi, N., Moriyama, H., Fujiwara, T., Fukumori, Y & Tanaka, N (1997) The 2.8 A structure of hydroxylamine oxidoreductase from a nitrifying chemolithotrophic bacterium, Nitrosomonas europaea Nat Struct Biol 4, 276–284.
8 Hendrich, M.P., Petasis, D., Arciero, D.M & Hooper, A.B (2001) Correlations of structure and electronic properties from EPR spectroscopy of hydroxylamine oxidoreductase J Am Chem Soc 123, 2997–3005.
9 Arciero, D.M., Hooper, A.B., Cai, M & Timkovitch, R (1993) Evidence for the structure of the active site heme P460 in hydro-xylamine oxidoreductase of Nitrosomonas europaea Biochemistry
32, 9370–9378.
10 Arciero, D.M & Hooper, A.B (1997) Evidence for a crosslink between c-heme and a lysine residue in cytochrome P460 of Nitrosomonas europaea FEBS Lett 410, 457–460.
11 Sayavedra-Soto, L.A., Hommes, N.G & Arp, D.A (1994) Char-acterization of the gene encoding hydroxylamine oxidoreductase
in Nitrosomonas europaea J Bacteriol 176, 504–510.
12 Bergmann, D J & Hooper, A.B (1994) The primary structure of cytochrome P460 of Nitrosomonas europaea: presence of a c-heme binding motif FEBS Lett 352, 324–326.
13 Bergmann, D.J., Zahn, J.A & DiSpirito, A.A (2000) Primary structure of cytochrome c¢ of Methylococcus capsulatus Bath: evidence of a phylogenetic link between P460 and c¢-type cyto-chromes Arch Microbiol 173, 29–34.
14 Moir, J.W.B (1999) Cytochrome c¢ from Paracoccus denitrificans: spectroscopic studies consistent with a role for the protein in nitric oxide metabolism Biochim Biophys Acta 1430, 65–72.
15 Tahirov, T.T., Misaki, S., Meyer, T.E., Cusanovitch, M.A., Higuchi, Y & Yasuoka, N (1996) High-resolution crystal struc-tures of two polymorphs of cytochrome c¢ from the purple phototrophic bacterium Rhodobacter capsulatus J Mol Biol 259, 467–479.
16 Bergmann, D.J., Zahn, J.A., Hooper, A.B & DiSpirito, A.A (1998) Cytochrome P460 genes from the methanotroph Methy-lococcus capsulatus Bath J Bacteriol 180, 6440–6445.
17 Cronin, C.N & McIntire, W.S (2000) Heterologous expression in Pseudomonas aeruginosa and purification of the 9.2-kDa c-type cytochrome subunit of p-cresol methylhydroxylase Protein Expr Purif 19, 74–83.
18 Sambrook, J., Frittsch, E.F & Maniatis, T (1989) Molecular Cloning: a Laboratory Manual, 2nd edn Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York.
19 Deng, W.P & Nicloff, J.A (1992) Site-directed mutagenesis of virtually any plasmid by eliminating a unique site Anal Biochem.
200, 81–88.
20 Chung, C.T., Niemala, S.L & Miller, R.H (1989) One-step pre-paration of competent Escherichia coli: Transfomation and sto-rage of bacterial cells in the same solution Proc Natl Acad Sci USA 86, 2172–2175.
21 Collins, M., Arciero, D.M & Hooper, A.B (1993) Optical spec-trophotometric resolution of the hemes of hydroxylamine oxido-reductase Heme quantitation and pH dependence of E m
J Biol Chem 268, 14655–14662.
Trang 722 Arciero, D.M., Peng, Q., Peterson, J & Hooper, A.B (1994)
Identification of axial ligands of cytochrome c552 from
Nitrosomonas europaea FEBS Lett 342, 217–220.
23 Laemmli, U.K (1970) Cleavage of structural proteins during
assembly of the head of bacteriophage T4 Nature 277, 680–685.
24 Schevchenko, A., Wilm, M., Vorn, O & Mann, M.M (1996)
Mass spectroscopy sequencing of proteins silver-stained in
poly-acrylamide gels Anal Chem 68, 850–858.
25 Zahn, J.A., Arciero, D.M., Hooper, A.B & DiSpirito, A.A (1996)
Cytochrome c¢ of Methylococcus capsulatus Bath Eur J Biochem.
240, 684–691.
26 Gilmour, R., Goodhew, C.F & Pettigrew, G.W (1991)
Cyto-chrome c¢ of Paracoccus dentrificans Biochim Biophys Acta 1059,
233–238.
27 Arciero, D.M & Hooper, A.B (1998) Consideration of a phlorin
structure for haem P-460 of hydroxylamine oxidoreductase and its
implications regarding reaction mechanism Biochem Soc Trans.
26, 385–389.
28 Einsle, O., Messerschmidt, A., Stach, P., Bourenkov, G.P., Bartunik, H.D., Hubner, R & Kroneck, M.H (1999) Struc-ture of cytochrome c nitrite reductase Nat Struct Biol 400, 476–480.
29 Wilkins, M.R., Gasteiger, E., Bairoch, A., Sanchez, J.-C., Williams, K.L., Appe, I.R.D & Hochstrasser, D.F (1998) Protein Identification and Analysis Tools in the Expasy Server.
In 2-D Proteome Analysis Protocols Humana Press, New Jersey (http://us.expasy.org/tools/peptide-mass.html)
30 Goodhew, C.F., Brown, K.R & Pettigrew, G.W (1986) Haem staining in gels, a useful tool in the study of bacterial c-type cytochromes Biochim Biophys Acta 852, 288–294.