From the Department of Biochemistry, University of
Oxford, South Parks Road, Oxford OX1 3QU, United Kingdom, the
Department of Biochemistry, Uppsala University, 75123 Uppsala,
Sweden, and the §§ Department of Biological
Sciences, University of Warwick,
Coventry CV4 7AL, United Kingdom
Received for publication, November 21, 2002, and in revised form, January 28, 2003
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ABSTRACT |
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The 1.4-Å crystal structure of the oxidized
state of a Y25S variant of cytochrome cd1
nitrite reductase from Paracoccus pantotrophus is
described. It shows that loss of Tyr25, a ligand via its
hydroxy group to the iron of the d1 heme in the
oxidized (as prepared) wild-type enzyme, does not result in a switch at
the c heme of the unusual bishistidinyl coordination to the
histidine/methionine coordination seen in other conformations of the
enzyme. The Ser25 side chain is seen in two positions in
the d1 heme pocket with relative occupancies of
~7:3, but in neither case is the hydroxy group bound to the iron
atom; instead, a sulfate ion from the crystallization solution is bound
between the Ser25 side chain and the heme iron. Unlike the
wild-type enzyme, the Y25S mutant is active as a reductase toward
nitrite, oxygen, and hydroxylamine without a reductive activation step.
It is concluded that Tyr25 is not essential for catalysis
of reduction of any substrate, but that the requirement for activation
by reduction of the wild-type enzyme is related to a requirement to
drive the dissociation of this residue from the active site. The Y25S
protein retains the d1 heme less well than the
wild-type protein, suggesting that the tyrosine residue has a role in
stabilizing the binding of this cofactor.
Cytochrome cd1 nitrite reductase is a
dimeric enzyme of the bacterial periplasm; it plays a key role in
denitrification, the respiratory reduction of nitrate to nitrogen gas.
The cytochrome c domain, carrying a heme that is attached to
the protein via two thioether bonds (Fig.
1), receives electrons from cupredoxins and c-type cytochrome donor proteins (1-4). The active site
on each subunit contains a specialized d1 heme
(Fig. 1) at which the physiological substrate, nitrite, binds and is
reduced. The d1 heme, found only in this class
of enzyme and bound in an eight-bladed
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-propeller structure (see
Fig. 2), is re-reduced in turn by electrons supplied by the heme of the
-helical c domain of the same subunit (see Fig. 2).
Release of the reaction product nitric oxide from the
d1 heme is not necessarily facile, as, in
general, nitric oxide binds very tightly to heme, especially the
ferrous state. Indeed, in several rapid reaction kinetic studies of
cytochrome cd1, retention of nitric oxide at the
d1 heme active site has been observed (e.g.
Refs. 5 and 6).
View larger version (17K):
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Fig. 1.
Schematic representations of the
structures of the heme in a c-type cytochrome and of
the d1 heme.
The crystal structures of cytochromes cd1 from two different species of bacteria, Paracoccus pantotrophus (formerly Thiosphaera pantotropha) (7) and Pseudomonas aeruginosa (8), have produced a number of surprises. Among these is the unusual ligation of the two types of heme in the oxidized (as prepared) form of the enzyme from P. pantotrophus. The heme of the c-type cytochrome center is bishistidinyl-coordinated, whereas the d1-type cytochrome center has a proximal histidine ligand but a distal tyrosine, residue 25. The latter is contributed by the cytochrome c domain of the protein and is only seven residues apart from, and on the same polypeptide loop as, histidine 17, which is one of the ligands to the iron atom of the c-type cytochrome center (7).
Upon reduction of crystals of the oxidized P. pantotrophus enzyme, histidine 17 was replaced as a ligand of the c heme iron by methionine 106 and tyrosine 25 dissociated from the d1 heme active site, leaving a five-coordinate iron (9). This suggested that ligand switching might be important in regulating the movement of electrons from the c to the d1 center and/or in providing a mechanism for displacing nitric oxide from the active site (7, 9). An alternative structure of the reduced cytochrome cd1 from this bacterium was subsequently obtained by crystallizing reduced enzyme. In this case, the relative positions of the c and d1 domains were different, but the ligand switching seen in the original crystal structures of the oxidized and reduced enzyme had still occurred (10). However, the "switched" ligands were retained after reoxidation of this new crystal form (10).
The presence in the P. pantotrophus enzyme of tyrosine 25 bound to the d1 heme and histidine 17 bound to the c heme was unexpected because neither of these residues is conserved in the cytochromes cd1 from other bacteria apart from the very closely related Paracoccus denitrificans. Accordingly, the structure of the oxidized cytochrome cd1 from P. aeruginosa (8) showed that the d1 heme was ligated differently from that in P. pantotrophus. In the former, a hydroxide was the distal ligand to the d1 heme iron, but with a hydrogen bond to a tyrosine residue (position 10) that was in no sense equivalent in the structure to tyrosine 25 in the P. pantotrophus enzyme. Tyrosine 10, which is not an essential residue (11), is not provided by the same subunit as that in which it is positioned close to the d1 heme iron, i.e. there is a domain swapping (8). The c heme also differed, with the equivalent of methionine 106 of the P. pantotrophus enzyme being a ligand in the oxidized P. aeruginosa enzyme, along with the histidine of the CXXCH motif that is characteristic of a c-type cytochrome. The coordination of the c-type cytochrome heme in the oxidized P. aeruginosa enzyme is thus equivalent to that in the reduced P. pantotrophus enzyme.
In solution, spectroscopic analysis is consistent with ligation of tyrosine to the d1 heme iron of the oxidized (as prepared) P. pantotrophus enzyme and has demonstrated the same bishistidinyl coordination of the c heme as seen in the crystal (12). However, it has recently been shown that a catalytically more active form of the protein is obtained if the enzyme is first reduced and then oxidized by one of its alternative substrates, hydroxylamine (13). This much more active (initially) oxidized form has His/Met coordination at the c heme (14), while visible absorption and EPR spectra indicate that tyrosine 25 is not bound to the d1 heme. Simple pre-reduction of the enzyme has also resulted in much higher kcat values than could be obtained with the oxidized (as prepared) enzyme for any of the electron donor proteins: cytochrome c550, pseudoazurin, or horse heart cytochrome c, in combination with any of the three known electron acceptors, nitrite, oxygen, or hydroxylamine (3, 13). Cytochrome cd1 can act as an oxidase; and in the early stages of this reaction an oxidized state of the protein with His/Met coordination at the c-type cytochrome center has been observed (10, 15), as was also the case for a form of the enzyme seen after a 1-electron oxidation of cytochrome cd1 by nitrite (5). His/Met coordination of the heme of the c domain in the oxidized state is also found in solution for both the semi-apo form of the protein that has lost the d1 heme (15) and the c domain expressed in isolation (16).
Although the combination of the observations on the catalytic activity of P. pantotrophus cytochrome cd1 and the structure of the P. aeruginosa enzyme suggests that bishistidinyl coordination at the c heme and tyrosine ligation at the d1 heme may not be significant in steady-state catalysis, there remain many unanswered questions about structure-function relationships for P. pantotrophus cytochrome cd1, particularly with respect to the role of tyrosine 25. For example, in a rapid reaction study starting from fully reduced enzyme and nitrite, nitric oxide was not released from the enzyme, suggesting that some conformational change was needed to trigger this event (Ref. 5 and see also Ref. 17). Structures of cytochrome cd1 from crystals that had first been reduced and then treated with nitrite showed tyrosine 25 apparently poised ready to displace a bound nitric oxide (9); the significance of this residue, but not necessarily its religation to the d1 heme iron, for this displacement has been indicated by calculations (17). The driving force for tyrosine 25 to ligate to the oxidized iron of the d1 heme is certainly large. Cytochrome cd1 from P. pantotrophus is exceptional in binding cyanide only when the d1 heme iron is in the ferrous state; cyanide, which normally binds very tightly to ferric hemes, is readily displaced by the tyrosine residue (18). Finally, there appears to be a very tight coupling between both the oxidation states and coordination at the c and d1 centers that is most clearly illustrated by the highly cooperative and hysteretic redox titration (19).
It is clear that many aspects of further progress in understanding cytochrome cd1 from P. pantotrophus will require the preparation of enzyme carrying mutations at the important residues that have been identified by crystallography. This is no easy task, as the very specialized d1 heme is only made by denitrifying bacteria, none of which is an ideal host for site-directed mutation studies. One approach is to make the semi-apoenzyme in vivo and then add the d1 heme in vitro. This has been done for the enzyme from P. aeruginosa, but it requires the preparation of large amounts of wild-type enzyme as a source of the d1 heme (e.g. Ref. 6). Production of an inactive cd1 in P. pantotrophus itself is problematic, as recent work on the expression of the nirS gene has shown that its transcription is activated by nitric oxide, the product of the reaction catalyzed by the enzyme (20). Furthermore, anaerobic conditions are needed for formation of cytochrome cd1, but use of nitrate as an anaerobic electron acceptor in the absence of an active cytochrome cd1 results in accumulation of toxic nitrite.
In view of these considerations, we sought in this work to identify a
variant form of P. pantotrophus cytochrome
cd1 that was still active and that might
therefore be expressed in P. pantotrophus itself. The recent
evidence from several types of study that tyrosine 25 might not be
essential for catalysis prompted us to investigate other residues that
might be tolerated at this position. Initially, serine was chosen
because the P. aeruginosa structure has the hydroxy of
tyrosine 10 at approximately the same distance from the
d1 heme iron as we estimated the serine hydroxy
would be in a putative Y25S mutant of the P. pantotrophus
enzyme. Thus, we anticipated that an active and stable folded form of
the latter enzyme, perhaps with a hydroxide lying between the serine
and the heme iron, analogous to the hydroxide that is the immediate ligand to the d1 heme of the P. aeruginosa enzyme, would be produced. This study reports that this
expectation of expression of a mutant form of P. pantotrophus cytochrome cd1 was realized
and the properties, including the structure, of the Y25S enzyme.
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EXPERIMENTAL PROCEDURES |
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Media, Strains, and Plasmids-- Escherichia coli and P. pantotrophus strains are listed in Table I. Luria broth or agar was routinely used for cultivation of these strains, which was carried out at 37 °C. Antibiotics were used at the following concentrations: ampicillin, 100 µg/ml; kanamycin, 50 µg/ml; gentamicin (700 units/mg) 20 µg/ml (or 100 µg/ml for E. coli); and rifampicin, 20 µg/ml. Minimal medium for growth of P. pantotrophus strains was prepared as described by Robertson and Kuenen (21); 20 mM succinate and 20 mM KNO3 were added where appropriate. DNA sequencing was performed using Applied Biosystems BigDye terminators as described by the manufacturer. DNA manipulations and transformations were carried out using standard procedures (22). Restriction and modifying enzymes (New England Biolabs Inc.) were used according to the manufacturer's protocols. Southern hybridizations were carried out in 6× SSC (22), 1× Denhardt's solution, and 50% formamide at 42 °C. The nirS gene from P. pantotrophus was mutated such that an EcoRI site was inserted immediately before the start codon, and a HindIII site was inserted immediately after the stop codon (23); the plasmid containing this construct is pEG10 (this has the nirS open reading frame as well as 125 bp upstream DNA (the nirS promoter), and ~600 bp downstream DNA (the start of the nirE gene)). This cassette was then used for further manipulations and mutagenesis of tyrosine 25 to serine. Site-directed mutagenesis was carried out according to Kunkel and Roberts (24) using the mutagenic primer 5'-GTCCAGCGAGGGCTCGCAGCGATTGTCGGTGCG-3'. The resultant mutated gene sequence for Y25S nirS was confirmed as correct over its entire length.
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Deletion of nirS from the Chromosome of P. pantotrophus-- A 2.7-kb PstI-SphI fragment containing the modified nirS gene was obtained by appropriate digestion of pEG10. This fragment was further digested with EcoRI and HindIII to remove the nirS coding sequence. The restriction sites were end-filled with T4 DNA polymerase, and the ahp (kanamycin resistance) gene from pRS551 (25), which was excised on a StuI fragment, was ligated to give pEG156. This nirS::ahp construct was then excised on a PstI-SphI fragment (the SphI site was end-filled with T4 DNA polymerase) and ligated with pJQ200KS (26) cut with SmaI and PstI restriction enzymes. This final plasmid, pEG181, was then used for gene replacement experiments exactly as described by Quandt and Hynes (26). The resulting strains were subjected to Southern blot hybridization analysis.
Construction of Plasmids for Production of NirS Derivatives-- A custom expression vector was constructed to facilitate expression of nirS and its derivatives in P. pantotrophus. The backbone of this vector was the 5-kb Csp45I-AatII restriction fragment from the broad host range vector pBBR1MCS (27). The gene encoding kanamycin resistance and fragments coding for the lacI gene, tac promoter, and rrnB terminators were amplified using PCR methodology from plasmids pHRP310 (28) and pMMB503EH (29), respectively. Restriction enzyme sites were added to the oligonucleotide primers such that the amplified products had the following constructions: Csp45I-lacI-NheI, XbaI-aph-SacII, and SacII-Ptac-MCS-rrn-AatII. Ligation of these fragments with the 5-kb Csp45I-AatII restriction fragment from pBBR1MCS generated plasmid pEG211, which thus carried the inducible tac promoter, with the lacI gene immediately downstream of the kanamycin gene, such that transcription from the aph gene also resulted in transcription of the lacI gene. A derivative of this plasmid, pEG276, consisted of the aac gene (coding for resistance to gentamycin) from pHRP309 (28) instead of the aph gene. A NsiI restriction site was also inserted immediately before the multiple cloning site, enabling the promoter driving overexpression (in this case, the rhn promoter from the rrnA operon of Rhodobacter sphaeroides) (30) to be changed easily. This promoter was obtained using primers rhnF (5'-CAACCGCGGCCTCCGGCGACGGGCCTG-3') and rhnR (5'-ACAATGCATCCGTCACCGTCGCCGTT-3') such that the former incorporated a SacII restriction site and the latter a NsiI restriction site. Thus, the amplified fragment could be transferred into a promoter-free expression vector to give pEG276.
Growth of Cells and Purification of the Y25S Protein-- The inoculum for the large-scale culture (as described above) was obtained by first inoculating 50 ml of Luria broth culture (in a 250-ml flask) with a colony from a Luria broth plate that had been freshly streaked with a glycerol stock 2 days earlier. This 50-ml culture was grown for ~15 h at 37 °C with shaking; 5-10 ml was then used to inoculate a 5-liter culture for enzyme preparation.
For the purification of Y25S cytochrome cd1, P. pantotrophus EG6202 cells carrying plasmid pEG607 were grown overnight (~17 h) at 37 °C in minimal medium with 20 mM succinate and 20 mM KNO3 under the selective pressure of 20 µg/ml gentamicin and 50 µg/ml kanamycin. It was crucial that diffusion of atmospheric oxygen into the culture was minimized. Hence, either fully filled 5-liter Erlenmeyer flasks with narrow necks and thus small surface area or 5-liter Duran bottles filled up to the neck with minimal medium were used to ensure that the culture was as anaerobic as possible. If a larger surface area of the culture was exposed, then the enhanced rate of oxygen transfer into the respiring culture resulted in formation of mainly semi-apoenzyme lacking the d1 heme.
Harvesting and purification procedures were similar to those used for the wild-type enzyme (31), i.e. a periplasmic preparation was first made using lysozyme. The periplasmic proteins were then loaded onto a DEAE-Sepharose fast flow column. Fractions (eluted by a gradient of 0.1-0.5 M NaCl) containing cytochrome cd1, as judged by visible absorption spectroscopy, were then applied, following addition of ammonium sulfate to 40% saturation, to a Poros HP2 hydrophobic chromatography column. This column was developed using a gradient of 40-0% ammonium sulfate. Fractions identified as containing cytochrome cd1 were finally subjected to gel filtration on Superdex 200 (Amersham Biosciences), which removed small amounts of impurities, to produce material for crystallization. This last step was omitted for samples used in kinetic and spectroscopic measurements.
Structure Determination-- Crystals of the Y25S mutant protein grew under conditions similar to those used for the wild-type enzyme (31), i.e. from a solution containing 10-20 mg/ml protein in the presence of 2.2-2.4 M ammonium sulfate and 50 mM potassium phosphate (pH 7.0). Data to a resolution of 1.4 Å were collected at 100 K using monochromatic x-rays at a wavelength of 0.934 Å on beam line I711 at the MAX Synchrotron (Lund, Sweden). The refined model of the oxidized protein was used for initial phasing (7). The phases were refined using the programs REFMAC (32) and CNS (33), and model building was done with the program O (34). The overall crystallographic R-factor for the final model was 15.9%, and the free R-factor was 17.5% (Table II).
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Other Methods--
Electrospray ionization mass spectrometry was
performed on a Micromass Bio-Q II-2S triple quadrupole atmospheric
pressure instrument equipped with an electrospray interface. Samples
were introduced via a loop injector as a solution (20 pmol/µl in 1:1 water acetonitrite and 1% formic acid) at a flow rate of 10 µl/min. Visible absorption spectra were obtained using a PerkinElmer Life Sciences 2 spectrophotometer. Steady-state kinetic assays were performed at 25 °C and pH 7.0 in 50 mM potassium
phosphate buffer as described previously (3) using cuprous P. pantotrophus pseudoazurin as electron donor. Data were analyzed as
described (3).
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RESULTS |
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Expression of the Protein--
Cells of P. pantotrophus
nirS carrying the Y25S variant of cytochrome
cd1 grew under anaerobic conditions at a similar
initial rate, but to a lower final cell density, as those carrying a
plasmid coding for the wild-type enzyme. However, although growth of
both wild-type cells and
nirS cells carrying the Y25S
mutation required anaerobic conditions for production of cytochrome
cd1, only the latter required extra precautions to
minimize entry of oxygen into the cultures (see "Experimental
Procedures"). The anaerobic cultures of cells containing the Y25S
variant of cytochrome cd1 evolved copious
amounts of gas, presumably nitrogen. This observation is indicative of
denitrification proceeding past the reduction of nitrite, which
accumulated transiently, to nitric oxide. Cells carrying the
deletion in the chromosomal copy of nirS, but without a
plasmid coding for an active enzyme, grew, without gassing, to only
restricted cell densities, and nitrite had accumulated in the medium at
the end of growth. Thus, it can be deduced that the Y25S variant enzyme
is sufficiently active in vivo to sustain growth of cells
under conditions that require reduction of nitrite.
Electrospray Mass Spectrometry-- The calculated mass (including the covalently bound c heme, but not the noncovalently bound d1 heme) of the Y25S variant of P. pantotrophus cytochrome cd1 is 63,022 Da. Electrospray mass spectrometry of the Y25S protein gave a value of 63,009 ± 1.5 Da. The interpretation of the difference between this value and the calculated value is that the N-terminal glutamine has cyclized with the elimination of a molecule of ammonia (17 Da). This conclusion was confirmed by failure to obtain an N-terminal amino acid sequence by Edman degradation. Such cyclization has been observed previously for wild-type P. pantotrophus cytochrome cd1 (7). Confirmation of this cyclization cannot be obtained from the crystal structure of the P. pantotrophus protein (wild-type or Y25S variant) because the first eight residues at the N terminus are disordered (Ref. 6 and see below). The 1.4-Å crystal structure of the Y25S variant (see below) clearly shows that there are no other amino acid changes throughout the protein, and the DNA sequence of the disordered part of the N-terminal arm region also indicates that it does not contain alterations in that region. Thus, the difference between the expected molecular mass (63,022 Da) and the determined value (63,009 Da) can be attributed with some confidence to the cyclization of the N-terminal residue.
Structure of the Y25S Mutant--
The structure of the Y25S mutant
of oxidized P. pantotrophus cytochrome
cd1 was determined to a resolution of 1.4 Å.
The overall structure is essentially identical to that of the wild-type
oxidized enzyme (Fig. 2) (7), with the
same small differences between the two subunits (called A and B) in the
dimer (root mean square difference = 0.1 Å when all C- atoms
are superimposed). There were additional differences in the proximity
of the mutated residue. The electron density shows unambiguously that
residue 25 has been mutated to serine. In both subunits, the serine 25 side chain adopts two conformations, which have approximate fractional
occupancies of 0.7 and 0.3 respectively (Fig.
3, A and B). Unlike
for tyrosine 25 in the wild-type enzyme, the hydroxy group of serine 25 in the Y25S variant is not directly bonded to the iron atom of the d1 heme. Electron density extending from the
d1 heme iron was interpreted as a sulfate ion
(Fig. 3, A and B), which was not within hydrogen
bonding distance of the serine hydroxy even in the dominant conformer
(70% occupancy), in which the ion and the side chain most closely
approach each other. As serine is not the natural residue at position
25, it is understandable that the hydroxy group, in either
conformation, is not within hydrogen bonding distance of a suitable
partner. The cumulative strength of the other interactions (Table
III) between the c and
d1 domains appears to account for the positions
of the serine side chain. It is notable that the sulfur atom of
methionine 409 is near to serine 25 and contributes to stabilizing the
predominant position of the side chain of the latter. The presence of
sulfate bound to the d1 heme is explicable on
the basis that sulfate was present at high concentrations during some
steps of the purification as well as in the solutions used for
crystallization. It may be recalled that sulfate provided a ligand for
the oxidized d1 heme in another structure of
cytochrome cd1 (10), and a sulfate ion heme
ligand was also reported in the structure of oxidized pentaheme (NrfA) nitrite reductase (35).
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The consequence for the structure of replacement of tyrosine 25 by
serine was remarkably localized to the immediate vicinity of the distal
side of the d1 heme. There was no effect on the position of the proximal ligand, histidine 200, or on the positions of
residues 24 and 26 (Fig. 3C). Indeed, surprisingly, the
conformation of the entire structurally ordered part of the N-terminal
arm (residues 9-48) (7) was unaffected by the mutation. All
noncovalent interactions, other than those associated with
Tyr25, between the N-terminal arm and both the c
and d1 domains are present in the mutant
structure. In particular, the unusual bishistidinyl coordination of the
c-type cytochrome center of the oxidized state of the
wild-type enzyme, with the two imidazole groups almost perpendicular to
each other, is retained in the Y25S mutant (Fig. 4).
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Visible Absorption Spectra--
The solution spectrum of the
oxidized Y25S protein differed dramatically from that of the wild-type
protein at the wavelengths characteristic of the
d1 heme. There was no band comparable to the
high-spin d1 heme absorption at 702 nm that is
found for the wild-type enzyme, but rather a broad absorbance with its
maximum at ~640 nm (Fig.
5a). This indicates that the
d1 heme is all low-spin in the Y25S variant, as
observed for wild-type P. aeruginosa and Pseudomonas
stutzeri cytochromes cd1 (12). Note that
for wild-type P. pantotrophus cytochrome
cd1, the His/Tyr-coordinated
d1 heme is in a high-spin/low-spin thermal
equilibrium around room temperature (12). In contrast, the absorption
spectrum of the reduced state of the Y25S variant protein was very
similar to that of reduced wild-type P. pantotrophus
cytochrome cd1, as illustrated in Fig. 5b. The characteristic (15) split -band at ~550 nm,
with the higher intensity at the shorter of the two wavelengths, was
retained. These data suggest that the reduced states of the two forms
of cytochrome cd1 are similar in terms of the
coordination and environment of both types of heme center in the
enzyme.
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Steady-state Kinetic Assays--
Assay with P. pantotrophus pseudoazurin as electron donor and nitrite, oxygen,
or hydroxylamine as acceptor showed that the activities were similar
for both the wild-type and Y25S mutant enzymes when both were
pretreated by reduction with dithionite, which activates the wild-type
enzyme (3). The kcat values (expressed per
monomer of cytochrome cd1) for nitrite, oxygen,
and hydroxylamine were 67, 6.2, and 7.7/s for the Y25S mutant and 68, 3.2, and 3.2/s for the wild-type enzyme, respectively. However, if a
pre-reduction step was omitted, then whereas the
kcat values for the wild-type enzyme were
reduced by 96% (3), there was no attenuation of the activity of the
mutant enzyme. The Km for nitrite was 63 µM for the Y25S protein, a similar value to that (71 µM) observed for the activated wild-type enzyme (3).
Because the absorption spectra of the reduced wild-type and Y25S
variant enzymes were so similar (Fig. 5b), the extinction
coefficient for reduced wild-type P. pantotrophus cytochrome
cd1 (323 mM cm1 at 418 nm) (15) was used to calculate concentrations of (and hence, kinetic
constants for) the Y25S enzyme.
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DISCUSSION |
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Significance of Tyrosine 25 Binding to the d1 Heme Iron-- When bishistidinyl coordination of the c-type cytochrome center and tyrosine 25 as a ligand to the d1 heme iron were first observed in crystals of oxidized cytochrome cd1 from P. pantotrophus, it was thought that the tyrosine residue may play a key role in displacing the reaction product, nitric oxide, from the d1 heme active site (7). Two structures of the reduced enzyme show that the tyrosine is displaced from the active site, consistent with opening up the d1 heme iron for binding of nitrite, and that the heme iron coordination at the c center has switched to histidine/methionine (10, 11). These and other studies (13, 15), along with the unusual hysteretic redox titration that demonstrated considerable cooperativity between the c and d1 centers (18), have all suggested that ligation of tyrosine 25 to the d1 heme is strongly coupled to the bishistidinyl coordination at the c heme center.
The properties of the Y25S mutant protein that are reported here show that the coordination of tyrosine 25 to the d1 heme is not required per se to trigger the adoption of His/His coordination at the c heme center. Even without Tyr25, there are sufficient other residues to make contacts (see below) to maintain the N-terminal arm structure that is characteristic of the crystalline state of the oxidized (as prepared) protein. This consideration explains why one crystal form seen previously with nitric oxide, rather than tyrosine 25, bound at the d1 heme could still have His/His coordination at the c-type center (9). In the latter structure, tyrosine 25 could be seen at the edge of the d1 heme-binding site.
The finding that, with a physiological electron donor as substrate, the Y25S protein is as active a nitrite reductase as the wild-type protein does not support the earlier proposal that Tyr25 plays a role in driving the dissociation of each nitric oxide from the active site of the enzyme through a steady-state cycle of direct coordination on to and release from the iron of the d1 heme (7). However, it is still possible, for example, that approach of tyrosine 25 toward the two active-site histidine residues (positions 345 and 388) plays a role in driving dissociation of nitric oxide (17) and that this role can be replicated by the hydroxy group of serine 25. In other words, as discussed elsewhere (17), the return of Tyr25 to a position between His345 and His388 may facilitate product (nitric oxide) release, but rebinding of Tyr25 to the oxidized iron of the d1 heme is bypassed during steady-state catalysis. As cytochromes cd1 from various sources have different ligands that can approach the d1 heme iron, a range of ligands (e.g. Tyr25 or Ser25) may interact with the conserved His345 and His388 so as to create an unfavorable orientation for nitric oxide and thus promote its dissociation (17).
The lack of requirement for a reductive pre-activation of the Y25S enzyme, in marked contrast to the wild-type enzyme (3), is intriguing. It is probable that the reduction potential of the bishistidinyl-coordinated heme is less positive than that of the electron donor proteins pseudoazurin and cytochrome c550 (19). However, this need not prevent electron transfer from these donors via the c domain heme center and on to the d1 heme active site if the overall reaction is exergonic. As discussed by Page et al. (36), there are many examples of uphill energetic steps in chains of electron carriers. Tyrosine is known to stabilize the ferric form of heme groups; thus, the absence in the Y25S mutant of the phenolic oxygen ligand to the d1 heme provided by tyrosine may raise the reduction potential of the d1 heme sufficiently to make it reducible by electrons originating from pseudoazurin. Hence, the triggering of dissociation of tyrosine 25 by reduction with dithionite that is needed to activate the wild-type enzyme (3) would not be required for the Y25S mutant. Alternatively (or additionally), the adoption of His/Met coordination of the c heme by the wild-type enzyme may be dictated, at least in part, by the requirement for concomitant removal of tyrosine 25 from its position blocking the entry of nitrite into the d1 heme active site (3). In the Y25S mutant, the serine is less effective at blocking the active site because it does not directly coordinate the d1 heme iron. Nitrite binding to the d1 heme iron and its reduction to nitric oxide (E'0 = +374 mV) provide a final step in the electron transport chain that is thermodynamically favored relative to the reduction potential of the electron donor proteins cytochrome c550 and pseudoazurin, for which E'0 is approximately +250 mV. It is clear that definitive answers to these issues will require substantial further work, which may also reveal whether P. pantotrophus cytochrome cd1 differs from or is similar to the counterpart in P. aeruginosa, where reduction of the d1 heme before the c heme, at least in the crystal, is linked to the conformational change that drives hydroxide from the active site (37).
The N-terminal Arm is an Important Structural Element in the Oxidized Monoclinic Structure-- A comparison of domain-domain interactions in the known conformers of P. pantotrophus cytochrome cd1 shows that the structure of the oxidized enzyme derived from monoclinic (P21) crystals has the largest domain-domain interface (Table III). The interface is stabilized by 19 or 20 hydrogen bonds and salt bridges between the N-terminal part of the enzyme and the d1 domain in subunits A and B, respectively. Of the 19 (20 in subunit B) interactions between the N-terminal part and the d1 domain, 10 (11 in subunit B) are mediated by the N-terminal arm (residues 9-48). Moreover, the N-terminal arm provides heme ligands to both the d1 and c hemes, and it thus constitutes an important structural element in the oxidized monoclinic structure. As can be seen in Table III, the replacement of Tyr25 by serine causes loss of only one interaction. It is tempting to conclude from this observation that the binding of Tyr25 to the the d1 heme iron is not critical for the conformation of the N-terminal arm that permits bishistidinyl coordination of the c heme. Rather, it appears possible that, in P. pantotrophus cytochrome cd1, it is reduction of the iron of the c heme that triggers the movement of this arm and the recruitment of Met106 as a ligand to the iron of this center. This would be consistent with the observation (e.g. Ref. 19) that, in general, oxidized (Fe(III)) heme is thermodynamically stabilized relative to Fe(II) heme by His/His coordination, whereas Fe(II) heme is relatively stabilized by His/Met coordination. However, Table III also shows that, in the more recently described tetragonal crystal form of P. pantotrophus cytochrome cd1 (10), there are far fewer contacts between the N- and C-terminal domains, in either the oxidized or reduced state. At present, the relative contributions to steady-state catalytic turnover of solution structures similar to those seen in the different crystal forms cannot be evaluated.
If neither tyrosine 25 ligation to the d1
heme nor bishistidinyl coordination of the c heme plays a
role in the catalytic activity of cytochrome cd1
from P. pantotrophus (3, 13-15), we are left with the task
of seeking an alternative explanation for the occurrence of these
structural features. As noted above, they are related to a significant
stabilization of the N-terminal domain in the oxidized crystal and can
also be deduced to occur in solution (12) on the assumption that their
presence correlates with bishistidinyl coordination at the c
heme center. Furthermore, the oxidized wild-type enzyme seems unable to
bind cyanide in solution; in common with the crystal, only the reduced
state of the enzyme binds this ligand in solution (18). Thus, the
strong driving force for adoption of the His/His-coordinated
c heme by the oxidized enzyme is not restricted to the
crystalline state. On the other hand, the recent demonstration, by a
combination of solution spectroscopies (13, 14), that a
His/Met-coordinated c heme conformer of the oxidized enzyme
can persist in solution for many minutes after the addition of
hydroxylamine to the reduced enzyme might suggest that the
bishistidinyl-coordinated c-type center and the
tyrosine-ligated d1 heme center are less
favored, at least in a kinetic sense, in solution than in the crystal. Cells carrying the Y25S mutation do not grow to the same density as
those carrying the same plasmid with the wild-type gene inserted. We
have also found that it is crucial to minimize the diffusion of
atmospheric oxygen into the culture producing the Y25S variant protein
(see "Experimental Procedures"). This finding is consistent with an
earlier suggestion (10) that, when a depletion of reducing equivalents
occurs in the periplasm, dioxygen or peroxides may react with an
open/unprotected d1 heme, creating harmful side reactions. Shutting off access to the d1 heme by
a return of Tyr25 to the iron would prevent the heme iron
from reacting with oxygen species in an uncontrolled manner. Such a
"shutoff" mechanism is not possible in the Y25S protein. We
found that an enhanced rate of oxygen transfer into the respiring
culture resulted in formation of mainly semi-apoenzyme, which lacks the
d1 heme. The Y25S enzyme obtained immediately
after cell breakage is contaminated to a greater extent than the
wild-type protein by semi-apoprotein. It therefore seems likely that
the presence of Tyr25 is advantageous because it stabilizes
the protein, particularly with respect to the initial insertion and/or
subsequent retention of the d1 heme.
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ACKNOWLEDGEMENTS |
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We are grateful to the staff of beam line I711 at the MAX Synchrotron for considerable assistance, Robin Aplin for the electrospray mass spectrometry analysis, and Tony Willis for N-terminal sequencing.
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FOOTNOTES |
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* This work was supported by Biotechnology and Biological Sciences Research Council Grant B11988, the Swedish Research Councils, and European Union Biotechnology Structural Biology Project BIO4 CT96-0281.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The atomic coordinates and the structure factors (code 1GQ1) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).
§ Both authors contributed equally to this work.
¶ Present address: Dept. of Molecular Biotechnology, Lundberg Laboratory, Chalmers University of Technology, Medicinaregatan 9C, SE-405 30 Göteborg, Sweden.
** Present address: AstraZeneca Research and Development, S-431 83 Mölndal, Sweden.
W. R. Miller Junior Research Fellow (St. Edmund Hall, Oxford).
¶¶ Royal Society University Research Fellow.
To whom correspondence should be addressed. Tel.:
44-1865-275-240; Fax: 44-1865-275-259; E-mail:
stuart.ferguson@bioch.ox.ac.uk.
Published, JBC Papers in Press, January 28, 2003, DOI 10.1074/jbc.M211886200
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