Activation of the cytochrome cd1 nitrite reductase from Paracoccus pantotrophus. Reaction of oxidized enzyme with substrate drives a ligand switch at heme c.

Abstract

Cytochromes cd(1) are dimeric bacterial nitrite reductases, which contain two hemes per monomer. On reduction of both hemes, the distal ligand of heme d(1) dissociates, creating a vacant coordination site accessible to substrate. Heme c, which transfers electrons from donor proteins into the active site, has histidine/methionine ligands except in the oxidized enzyme from Paracoccus pantotrophus where both ligands are histidine. During reduction of this enzyme, Tyr(25) dissociates from the distal side of heme d(1), and one heme c ligand is replaced by methionine. Activity is associated with histidine/methionine coordination at heme c, and it is believed that P. pantotrophus cytochrome cd(1) is unreactive toward substrate without reductive activation. However, we report here that the oxidized enzyme will react with nitrite to yield a novel species in which heme d(1) is EPR-silent. Magnetic circular dichroism studies indicate that heme d(1) is low-spin Fe(III) but EPR-silent as a result of spin coupling to a radical species formed during the reaction with nitrite. This reaction drives the switch to histidine/methionine ligation at Fe(III) heme c. Thus the enzyme is activated by exposure to its physiological substrate without the necessity of passing through the reduced state. This reactivity toward nitrite is also observed for oxidized cytochrome cd(1) from Pseudomonas stutzeri suggesting a more general involvement of the EPR-silent Fe(III) heme d(1) species in nitrite reduction.

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Activation of the Cytochrome cd1Nitrite Reductase from
Paracoccuspantotrophus
REACTIONOFOXIDIZEDENZYMEWITHSUBSTRATEDRIVESALIGANDSWITCHATHEMEc*
Receivedforpublication,February9,2007,andinrevisedform,July5,2007 Published,JBCPapersinPress,July10,2007,DOI10.1074/jbc.M701242200
Jessica H. van Wonderen‡, Christopher Knight‡, Vasily S. Oganesyan‡, Simon J. George§, Walter G. Zumft¶,
and Myles R. Cheesman‡1
Fromthe‡CentreforMetalloproteinSpectroscopyandBiology,SchoolofChemicalSciencesandPharmacy,Universityof
EastAnglia,NorwichNR4 7TJ,UnitedKingdom,¶InstituteofAppliedBiosciences,DivisionofMolecularMicrobiology,
UniversityofKarlsruhe,PF6980,76128Karlsruhe,Germany,and§PhysicalBiosciencesDivision,LawrenceBerkeley
Laboratory,Berkeley, California 94720
Cytochromes cd1are dimeric bacterial nitrite reductases,
which contain two hemes per monomer. On reduction of both
hemes,thedistalligandofhemed1dissociates,creatingavacant
coordination site accessible to substrate. Heme c, which trans-
fers electrons from donor proteins into the active site, has histi-
dine/methionine ligands except in the oxidized enzyme from
Paracoccuspantotrophuswherebothligandsarehistidine.Dur-
ing reduction of this enzyme, Tyr25dissociates from the distal
side of heme d1, and one heme c ligand is replaced by methio-
nine.Activityisassociatedwithhistidine/methioninecoordina-
tion at heme c, and it is believed that P. pantotrophus cyto-
chrome cd1is unreactive toward substrate without reductive
activation. However, we report here that the oxidized enzyme
willreactwithnitritetoyieldanovelspeciesinwhichhemed1is
EPR-silent. Magnetic circular dichroism studies indicate that
heme d1is low-spin FeIIIbut EPR-silent as a result of spin cou-
plingtoaradicalspeciesformedduringthereactionwithnitrite.
This reaction drives the switch to histidine/methionine ligation
at FeIIIheme c. Thus the enzyme is activated by exposure to its
physiologicalsubstratewithoutthenecessityofpassingthrough
the reduced state. This reactivity toward nitrite is also observed
for oxidized cytochrome cd1from Pseudomonas stutzeri sug-
gestingamoregeneralinvolvementoftheEPR-silentFeIIIheme
d1species in nitrite reduction.
Cytochrome cd1(cd1)2is a soluble homodimeric enzyme
involved in bacterial respiratory denitrification (1–3). It cata-
lyzes the one-electron reduction of nitrite ion (NO2
oxide (NO).
?) to nitric
NO2
?? 2H?? e?3 NO ? H2O
(Eq. 1)
The enzyme monomer comprises two distinct domains, one of
which binds heme c, the other heme d1(4). Heme c transfers
electrons from donor proteins to the active site heme d1, an
iron-dioxoisobacteriochlorin unique to this class of enzyme. In
the crystal structure of the aerobically isolated (“oxidized”) cd1
from Paracoccus pantotrophus (Pp), each heme is in the FeIII
state with two protein-derived axial ligands, His17and His69to
heme c and His200and Tyr25to heme d1(see Scheme 1). MCD
and EPR spectroscopy confirmed that these are the ligands in
solution (5). Crystallography further showed that reduction of
both hemes to the FeIIstate triggers a conformational change,
whichresultsinthereplacementofthehemecligandHis17with
Met106and the dissociation of Tyr25from heme d1(6), thus
creating a vacant coordination site at which substrate can bind
(shownintoplineofScheme1).Bothdissociatinghemeligands
are provided by a 7-residue c-domain loop, which extends into
the d1domain (6, 7). The switch to His/Met ligation signifi-
cantly raises the reduction potential of heme c, more closely
matching the donor proteins cytochrome c550and pseudoa-
zurin (8–13). This led to the speculation that this facilitates
electrontransfer(8)butalowhemecpotentialisnotstrictlyan
obstacle because the overall electron transfer from donors to
NO2
the ligand switch is therefore unclear. Other cytochromes cd1
have His/Met liganded hemes c and lack a residue correspond-
ing to His17. In these species, reduction appears sufficient to
triggerdissociationofthedistalhemed1ligandwithoutacoor-
dinated ligand switch at heme c (10, 15–17).
Reoxidation of reduced cytochromes cd1using NO2
mixture of half-reduced species containing stable FeII-nitrosyl
heme d1(18–21), although a recent report described rapid NO
dissociation from the fully reduced enzyme (22). Reoxidation
of fully reduced cd1using dioxygen gives rise to oxoferryl
(FeIV?O) heme d1and a protein radical (23). Hydroxylamine,
however, is a convenient two-electron non-physiological sub-
strate,whichstoichiometricallyreoxidizesbothhemesinasin-
gle turnover (11, 12, 24, 25).
?is thermodynamically favorable (13, 14). The purpose of
?yields a
H2N-OH ? 2H?? 2e?3 NH3? H2O
(Eq. 2)
* This work was supported by the Biotechnology and Biosciences Research
Council (BBSRC) studentship award 02A1B08117 (to J. v. W.), an Engineer-
ingandPhysicalSciencesResearchCouncil(EPSRC)advancefellowship(to
V. S. O.),andaWellcomeTrustawardfromtheJointInfrastructureFundfor
equipment.Thecostsofpublicationofthisarticleweredefrayedinpartby
the payment of page charges. This article must therefore be hereby
marked“advertisement”inaccordancewith18U.S.C.Section1734solelyto
indicate this fact.
1To whom correspondence should be addressed: School of Chemical Sciences
and Pharmacy, University of East Anglia, Norwich NR47TJ, United Kingdom.
Tel.:01603592028;Fax:01603582023;E-mail:m.cheesman@uea.ac.uk.
2The abbreviations used are: cd1, cytochrome cd1; Pp, Paracoccus pantotro-
phus; Pa, Pseudomonas aeruginosa; Ps, Pseudomonas stutzeri strain ZoBell
(ATCC 14405); CT, charge-transfer; MCD, magnetic circular dichroism; NIR,
near-infrared; BTP, 1,3-bis(tris(hydroxymethyl)methylamine)propane; RD-
MCD, ratio data method of MCD spectroscopy; H, magnetic field; T, tesla.
THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 282, NO. 38, pp. 28207–28215, September 21, 2007
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With Pp cd1, the product of this reaction, freeze-trapped after
?10ms,displaysFeIIIhemecEPRgvaluesdifferentfromthose
of the oxidized enzyme (11). Because these g values are almost
identical to those observed for the His/Met liganded heme c in
other cytochromes cd1(see Table 1), it was suggested that the
Pp enzyme has been trapped in a transient form (cd1* on
Scheme 1) in which heme c retains the coordination normally
associated with the reduced state (11, 12). Unambiguous iden-
tification of a His/Met ligand pair by MCD spectroscopy con-
firmed this to be the case (12). The transient cd1* reverts to the
oxidized conformation with His/His heme c over a period of
?20 min (11). Until this reversion has occurred, the enzyme is
capable of NO2
donor pseudoazurin (11). It is reported that for full activity,
oxidized Pp cd1requires preactivation by chemical reduction
(11, 13). Consequently, His/Met ligated heme c is presumed to
be a prerequisite of activity, and it has been proposed that the
oxidized enzyme is a resting form bypassed in catalysis as the
enzyme cycles rapidly compared with the rate of reversion to
the His/His form (13, 26). The oxidized state would therefore
not exist in vivo without an appropriate reactivation mecha-
nism (8, 12, 26). The only candidate suggested to date is reduc-
tion by NapC, a tetraheme protein with reduction potentials
ranging down to ?235 mV (27, 28).
Theintermediatecd1*differsfromoxidizedPpcd1inthatitis
reported to bind NO2
hydroxylamine reoxidation. The absorption spectrum of this
putative NO2
hemed1(29).ItwasconcludedthatNO2
ofFeIIIhemed1beforetherelativelyslowrebindingofTyr25can
occur. However, several observations make it unlikely that the
product(hereaftercd1*-X)isthesimpleNO2
d1species depicted in the lower left of Scheme 1. The long
timescaleofformationisnotconsistentwithaprocess,whichis
preventing the reversion to the oxidized state (29). The EPR
spectrum contained no signals characteristic of low-spin FeIII
heme d1. A single species was observed with similar g values to
those of His/Met heme c in cd1* (29) (Table 1). It was proposed
that NO2
Tyr25and preventing the conformational change to His/His
?reduction using the physiological electron
?immediately following its formation by
?-bound form is characteristic of low-spin FeIII
?bindstothedistalside
?-boundFeIIIheme
?bound at heme d1was blocking the rebinding of
ligation at FeIIIheme c. This is not strictly proven because
ligand orientation, not identity, is the primary determinant of
the form of the EPR spectrum (30). Rotation of the histidine
ligands from the perpendicular conformation observed in oxi-
dized cd1could yield the same g values. The work described
here was therefore undertaken with two primary objectives: to
determinetheidentityoftheligandstohemecincd1*-Xandto
provide an explanation for the EPR silence of the heme d1.
EXPERIMENTAL PROCEDURES
Purification of cd1—Samples of cd1from Pp and Pseudomo-
nas stutzeri (Ps) strain ZoBell (ATCC 14405) were purified
according to published methods (10, 31). Initially, samples of
the Pp cd1*-X were prepared according to reported methods
(11, 29): oxidized Pp cd1was reduced in an anaerobic hood
(FaircrestEngineering;operatingat?1ppmO2inanN2atmo-
sphere) using aliquots of buffered solutions of sodium dithion-
ite; excess reductant was removed by chromatography on a
PD-10 desalting column; the enzyme was reoxidized using ali-
quots of buffered hydroxylamine solution. Aliquots of buffered
potassium nitrite solutions, freshly prepared, were added
within 10–15 s of this reoxidation. Assays of nitrite reductase
activity were performed following published methods (13)
using reduced equine cytochrome c as electron donor.
EPR spectra were measured with a Bruker ER300D spec-
trometer fitted with a dual mode cavity type ER4116DM inter-
faced to an ELEXSYS computer control system (Bruker Ana-
lytische Messtechnik GmBH) and equipped with a variable
temperature cryostat and liquid helium transfer line (Oxford
Instruments). EPR simulations were performed using the
BrukerprogramWINEPRSimFonia(v1.25).MCDspectrawere
recorded on JASCO circular dichrographs models J810 and
J730fortheUV-visibleandnear-infrared(NIR)regions,respec-
tively, used in conjunction with an Oxford Instruments SM4
split-coilsuperconductingsolenoidcapableofgeneratingmag-
netic fields of up to 5 tesla.
The intensities of spectra presented are referred to concen-
trations of cd1monomer, calculated using the Soret absorption
intensityoftheoxidizedenzymesandextinctioncoefficientsof
? ? 141 mM?1cm?1for the Ps enzyme (32) and ? ? 148 mM?1
cm?1for the Pp enzyme (10). Samples for MCD spectroscopy
were prepared in deuterium oxide solutions containing 50 mM
BTP buffer at pH* ? 6.5 to which equivalent volumes of glyc-
erol had been added as glassing agent (33). pH* is the apparent
pHoftheD2O-basedsolutionsmeasuredusingastandardglass
pH electrode.
RESULTS
Preparation and Absorption Spectra of cd1*-X—Samples of
the Pp cd1*-X species were prepared anaerobically at pH ? 7.0
according to the methods described by Allen et al. (29). After
2-h incubation, the same type of absorption and EPR spectro-
scopic changes were observed. However, identical results were
obtainedduringcontrolexperimentsinwhichoxidizedcd1was
incubated with the same concentration (5 mM) of NO2
was the case whether the incubation was performed anaerobi-
cally or aerobically, but at pH 7.0, the reaction still required
severalhourstogotocompletion.Furthercontrolexperiments
?. This
SCHEME 1. The two-electron reoxidation of reduced Pp cd1by hydroxyl-
amine followed by slow reversion to the oxidized state (within box) and
the proposed trapping of cd1* by NO2
?.
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revealed that the rate of this reaction of resting enzyme with
NO2
lower pH. Although there are some variations in the rate
between preparations, at pH 6.5, the product is fully formed in
5–10 min but at pH 9.0 negligible reaction was observed over
24 h. The substrate itself (pKa3.35) is fully deprotonated over
the pH range investigated, and the pH dependence may be a
characteristic of NO2
No significant changes in the absorption spectrum of the oxi-
dizedenzymewereobservedoverthepHrange5.5–9.0.Assays
of NO2
firmed that cd1*-X prepared by this method is catalytically
active,asisreportedforthespeciesformedbyNO2
enzyme reoxidized with hydroxylamine (29). All samples used
for the spectroscopic characterization of cd1*-X described in
this work were prepared in D2O-based buffers at pH* 6.5 by
aerobic additions of buffered solutions of potassium nitrite.
The absorption spectra of oxidized Pp cd1and of cd1*-X are
shown in Fig. 1a. In oxidized Pp cd1at room temperature, FeIII
heme d1exists in a thermal equilibrium between a low-spin
ground state (S ?1⁄2) and an excited high-spin state (S ?5⁄2),
which give rise to absorption bands at 644 and 702 nm, respec-
tively (5). The cd1*-X spectrum lacks the high-spin FeIIIheme
d1band at 702 nm, but the low-spin band has increased in
intensity and is blue-shifted to 631 nm. Low-spin FeIIIheme c
bands between 500 and 600 nm are present in both forms. It
therefore appears that all FeIIIheme d1is in the low-spin state
following reaction with NO2
cd1*-X is very similar to those of the oxidized cytochromes cd1
?was strongly pH-dependent, increasing significantly at
?binding and/or the subsequent reaction.
?reductase activity using published methods (13) con-
?additionto
?. The absorption spectrum of Pp
from Pa and Ps, enzymes in which the hemed1is fully low-spin
at all temperatures (5, 19). This reactivity of oxidized Pp cd1is
unexpected given that it is generally accepted that this state
does not interact with NO2
the Pp enzyme is unique among known cytochromes cd1, and
this raises the question as to whether the ability to react with
NO2
Ps also reacts with NO2
treated with NO2
absorption band undergoes a small shift from 643 to 640 nm
(Fig. 1b). However, in this enzyme, formation of a cd1*-X spe-
cies, which contained low-spin FeIIIheme d1would involve no
change of spin-state at either heme. Less significant absorption
changes would therefore result. That, these small changes do
indicate the formation a cd1*-X, species is confirmed by EPR
and MCD spectroscopic studies described below.
EPRSpectraofcd1*-X—Fig.2showsthe10KX-bandperpen-
dicularmodeEPRspectraoftheoxidizedcytochromescd1from
Pp and Ps, and of the species formed following treatment of
eachenzymewithNO2
mode for any of these samples. Heme c in oxidized Pp cd1(Fig.
2a) gives rise to a “large gmax” spectrum with a gzfeature at
?3.06. This is one of two limiting types of EPR observed for
low-spin FeIIIc-type hemes (5, 30). The other two g values are
often difficult to detect but spectral simulations, presented
below,showthatbroadgyfeaturesarepresentintheg?2.2–2.3
?(34). The heme c ligand switch of
?inthismannerisalsounique.Wefoundthatthecd1from
?at pH 6.5. When the Ps enzyme is
?under identical conditions, the heme d1
?.Nosignalsweredetectedintheparallel
FIGURE1.Electronicabsorptionspectraofcd1fromPp(a)andPs(b)inthe
oxidized state (– – –) and after the addition of potassium nitrite (——).
Samples were in 50 mM BTP, D2O, pH* 6.5. Where present, NO2
tration was 5 mM.
?ion concen-
FIGURE2.X-bandEPRspectrashowingtheeffectofNO2
oxidized Pp (a), ?NO2
mM BTP, D2O, pH* 6.5. Where present, NO2
trometerparameters:microwavepower,2milliwatt;modulation,1millitesla;
temperature, 10 K.
?additiontocd1:
?(d). Samples were in 50
?concentration was 5 mM. Spec-
?(b), oxidized Ps (c), ?NO2
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region.HemecofoxidizedPscd1givesrisetoanEPRspectrum
of the second limiting type. This “rhombic” spectrum (Fig. 2c)
comprisesthreedetectablefeaturesatg?2.99,2.29,1.61.Note
that, although the hemes c in these two enzymes have different
axial ligation, it is not this which leads to the different EPR
spectra. The occurrence of a large gmaxor rhombic spectrum is
dictatedprimarilybyligandorientationandnotligandidentity.
ThusbothligandsetscouldgiverisetoeithertypeofEPRspec-
trum.InthecaseoftheHis/His-coordinatedhemecofoxidized
Pp cd1, the observation of a large gmaxspectrum is consistent
with the perpendicular ligand arrangement observed in the
crystal structure (4, 30).
At 10 K, only the low-spin state of FeIIIheme d1in oxidized
Pp cd1is significantly populated, and minor traces of the high-
spin state are observed at g values of 6.86 and 4.99 (5). The
low-spin FeIIIstate of heme d1gives rise to EPR spectra, which
are unlike either of the limiting cases described for heme c,
having narrow features and low g value anisotropy (5). These
are observed in the oxidized cd1spectra with g values of 2.52,
2.19, 1.84 for Pp and 2.58, 2.44, 1.87 for Ps. After addition of
NO2
acteristic low-spin FeIIIheme d1features, although this state of
d1is apparent in the absorption spectra (Fig. 1). The Ps cd1*-X
EPR spectrum (Fig. 2d) retains the signals which were assigned
to heme c in the oxidized state, and it is reasonable to assume
thatthatthesegvaluesdoindicateHis/Metligationbecausethe
cd1from this species does not exhibit ligand switching. For
NO2
c has been replaced by a rhombic spectrum, with g values of
2.93, 2.32, 1.42 as reported by Allen et al. (29), who proposed
that these features originate from FeIIIheme c with His/Met
ligation.Thevalidityofthisassignmentwillbeaddressedbelow
usingMCDspectroscopy.Ithasnotpreviouslybeenshownthat
this EPR spectrum accounts for a single heme, although, given
its unusual electronic ground state, low-spin FeIIIheme d1is
unlikely to give rise to these signals (5, 30, 35). The EPR signals
assigned to heme c in the cd1*-X forms (Fig. 2, b and d) were
quantitated by double integration of the spectra and compari-
son to a CuII-EDTA spin standard using established methods
(36).Eachrepresentsasinglelow-spinFeIIIhemepermonomer
confirming that heme d1is EPR-silent.
In the EPR spectra of the oxidized enzymes (Fig. 2), several
features appear to have incompletely resolved structure sug-
gesting minor heterogeneities at each heme. These features are
preparation-dependentandofunknownorigin.Becausecd1isa
dimeric protein and we have identified a form in which both
hemes are apparently in the low-spin FeIIIstate but heme d1is
EPR-silent, this raises the possibility that oxidized samples
actually include substoichiometric levels of cd1*-X so that in
some dimers one monomer contains an EPR-silent heme d1.
This would constitute a heterogeneity, which could be respon-
sible for the EPR splitting. However, if this was so then the
contribution of heme d1to the overall EPR envelope would be
less than that of heme c. To the best of our knowledge, it has
never been determined that the EPR signals arising from heme
c and heme d1represent equivalent concentrations of heme.
This has therefore been tested by simulating the EPR spectrum
of each oxidized enzyme. The spectra for simulation were
?, the EPR spectra (Fig. 2, b and d) are devoid of the char-
?-treated Pp cd1(Fig. 2b), the large gmaxspectrum of heme
recorded under non-saturating conditions. This required
recordingatatemperatureof20K,andbecausethespin-lattice
relaxationofhemed1isslowerthanthatofhemec,amicrowave
power of 0.64 milliwatt. The microwave power-dependence of
the two hemes at 10 and 20 K is shown in Fig. 3. The spectrum
of each oxidized enzyme was simulated using contributions
from two low-spin FeIIIheme c species (caand cb) and two
low-spin FeIIIheme d1species (daand db). The position and g
value for the low-field feature of each simulated species is also
indicated on the simulations in Fig. 4. All the g values used in
these simulations are included in Table 1, where the weighting
for each species is shown in brackets as percentage figures cal-
FIGURE 3. The EPR power saturation characteristics of hemes c and d1in
oxidized Pp cd1, measured by the intensity of the low-field features: gz
for heme c at 10 (‚) and 20 K (E); gxfor heme d1at 10 (Œ) and 20 K (F).
FIGURE 4. Experimental (——) and simulated (????) X-band EPR spectra of
oxidized cd1from Pp (a) and Ps (b). Samples were in 50 mM BTP, D2O, pH*
7.0.Spectrometerparameterswere:microwavepower,0.64milliwatt;modu-
lation,1millitesla;temperature,20K.Experimentalandsimulatedspectraare
offset for clarity.
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culatedaftersettingthetotalhemecto1.00ineachcase.Forthe
Ps enzyme, the heme d1contributions to the simulations rep-
resentonly?2/3ofthoseofhemec,whichwouldbeconsistent
withthepresenceofsomeEPR-silenthemed1.Howeverforthe
Pp enzyme, which shows a more pronounced EPR heterogene-
ity,theratiooftotalhemed1tototalhemecis0.99.Thusinthe
Pp enzyme at least, both hemes are fully EPR detectable, and
cd1*-X is not the cause of the observed EPR heterogeneities.
Furthermore, it is not apparent from these data that the heter-
ogeneities at the two hemes are correlated because in none of
the spectra does the percentage distribution between caand cb
match that between daand db.
NIR-MCD Spectra: the Heme c Ligands in cd1*-X—Heme c
givesrisetoalmostidenticalrhombicEPRspectraincd1*andin
cd1*-X(Table1andFig.2),butasexplainedabove,thisdoesnot
necessarily indicate that heme c also has His/Met axial ligation
incd1*-Xbecausethesegvaluescouldalsoresultfromachange
intherelativeorientationoftwohistidineligandsfromperpen-
dicular to parallel. As was reported for cd1* (12), the identity of
thehemecligandsincd1*-Xcanbeunambiguouslydetermined
usingMCDspectroscopyintheNIRregion(700–2000nm).At
these wavelengths, low-spin FeIIIhemes give rise to a charac-
teristic porphyrin-to-FeIIIcharge-transfer (CT) transition, the
exact energy of which is diagnostic of the axial ligands to the
heme(37,38).However,fortheunusual(dzx,dyz)2(dxy)1ground
stateoflow-spinFeIIIhemed1,thebandissymmetryforbidden
andhasnotbeendetected(5,35).Consequently,thetransitions
observed in this region are solely due to low-spin FeIIIheme c.
NIR-MCDspectra,recordedat4.2K,areshowninFig.5forthe
four forms of cd1reported in Figs. 1 and 2. The hemes c in
oxidized Pp cd1and oxidized Ps cd1give rise to CT bands with
peaks at approximately 1550 and 1775 nm, wavelengths diag-
nostic of His/His and His/Met coordination, respectively, as is
previouslyreported(5).ForPscd1*-X,thebandremainsat1775
nm confirming retention of His/Met ligands. However, on for-
mation of cd1*-X in the Pp enzyme, the CT band is red-shifted
from 1550 to 1795 nm proving that a ligand switch to His/Met
at heme c has occurred.
The Electronic State of Heme d1in cd1*-X—This work has
establishedthatthelow-spinFeIIIhemecincd1*-XhasHis/Met
ligationandso,inthecaseofthePpenzyme,aligandswitchhas
been triggered in the oxidized enzyme by the reaction with
NO2
appears to be low-spin FeIIIon the basis of absorption spectra
but is EPR-silent. The electronic and magnetic properties of
thiscenterhavethereforebeeninvestigatedusingvariablemag-
netic field variable temperature MCD spectroscopic methods.
The UV-visible region MCD spectrum of a heme contains an
extremely detailed pattern of electronic bands, and it is well
establishedforb-andc-typehemesthatthispatternisdiagnos-
tic of the spin and oxidation state of the iron, including the
EPR-silent FeIIstates (38). Although heme d1is unique to this
class of enzyme, and the available data is limited, it is clear that
heme d1MCD spectra are also sensitive to changes in spin and
oxidation state (5, 32, 39–42). The UV region MCD of cd1is
dominatedbyanintensehemecSoretbandbutatvisiblewave-
lengths, transitions of both hemes are readily observed. Fig. 6a
shows MCD spectra of the cd1*-X form of the Pp enzyme
recorded in this wavelength region using a magnetic field of 5
?. The nature of heme d1in cd1*-X is complex in that it
TABLE1
EPR g values of low-spin ferric hemes c and d1in cytochromes cd1
Heme c gz, gy, gx
Heme d1gx, gy, gz
a
Refs.
P. pantotrophus
Oxidized
Simulations
3.04 (2)b, —c,- –
ca3.140, 2.235, 1.060 (84%)d
cb2.980, 2.280, 1.430 (16%)
2.94, 2.33, 1.40
2.93 (1), 2.32, 1.40 (2)
2.51 (1), 2.20 (1), 1.85 (1)
da2.588, 2.217, 1.863 (72%)
db2.533, 2.217, 1.837 (29%)
2.67, 2.53 (1), —
Not observed
5, 11, 23
This work
cd1*
cd1*-X
P. stutzeri
Oxidized
Simulations
11
29, This work
2.96 (4), 2.27 (3), 1.62 (5)
ca2.997, 2.275, 1.400 (79%)
cb2.841, 2.340, 1.560 (21%)
2.99, 2.29, —
2.54 (4), 2.40 (5), 1.85 (2)
da2.583, 2.440, 1.614 (55%)
db2.466, 2.266, 1.867 (10%)
Not observed
5, 57
This work
cd1*-X
P. aeruginosa
Oxidized
cd1*-X
T. denitrificans
Oxidized
This work
2.97 (5), 2.25 (1), 1.4
2.95, 2.30, 1.42
2.52 (1), 2.43, 1.7 (6)
Not observed
18, 32, 39, 45
45
3.60, — ,—2.52, 2.44 (1), 1.70 58, 59
aThe order of g values is reversed in the unusual (dxz,yz)4(dxy)1ground state of low-spin FeIIIheme d1(5, 30).
bNumbers in parentheses represent the variation in the second decimal place in g values reported for each feature. This includes spread due to a 2-fold heterogeneity often
observed for cd1EPR features.
c—, g-values not determined from the spectrum.
dPercentages in parentheses are the weightings used for each species in the simulations of Fig. 4.
FIGURE5.Low-temperatureNIR-MCDspectraofcd1fromPpandPsinthe
oxidizedandcd1*-Xstates.Ppoxidized(– – –);Ppcd1*-X(——);Psoxidized
(????); Ps cd1*-X (–??–??–). Samples were in 1:1 (v:v) 50 mM BTP, D2O, pH* 6.5/
glycerol. Spectra were recorded at a temperature of 4.2 K with a magnetic
field of 5 tesla.
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teslaandatthreetemperatures,1.7,4.2,and10K.Allbandsare
strongly temperature-dependent indicating that they arise
fromparamagneticcenters.TheMCDbandpatternoflow-spin
FeIIIc-type hemes is well known (38). The major transitions
attributable to low-spin FeIIIheme c are therefore readily
assignedandareindicatedintheupperpartofFig.6abyblocks,
which are offset vertically to illustrate bands of positive and
negative intensity. Thus, in the region 520–590 nm, the nega-
tive feature at 557 nm and the two positive bands to either side
are solely due to low-spin FeIIIheme c. Blocks in the lower part
of Fig. 6a illustrate the positions and signs of transitions, which
havepreviouslybeenobservedforhemed1inthelow-spinFeIII
state (5, 32, 39, 40, 42). Transitions from both hemes overlap at
450–520 nm but features in the 590–700 nm region are pure
heme d1transitions.3The overall pattern of bands in the MCD
spectrum here is virtually identical to that previously reported
for oxidized cd1from Ps, in which both hemes are already
entirely low-spin FeIII(5). These spectra therefore further sup-
port the conclusion that in cd1*-X, heme d1is in the low-spin
FeIIIstate and is responsible for paramagnetic MCD intensity
despite the fact that it does not give rise to a detectable EPR
spectrum at X-band.
Thereisnoprecedentforanisolatedlow-spinFeIIIhemenot
giving rise to a detectable EPR spectrum but there are cases of
FeIIIhemes being rendered EPR-silent by a spin-spin interac-
tionwithasecondparamagneticcenter.Forexample,theheme
at the active site of bacterial nitric oxide reductase gives rise to
MCD bands characteristic of the high-spin FeIIIstate but it is
EPR-silentasaresultofspin-couplingwithanearbynon-heme
FeIIIiron (43). Such an explanation that FeIIIheme d1in cd1*-X
is spin-coupled to a second paramagnet would reconcile the
EPR and MCD data, and this possibility was investigated by
further MCD measurements.
The pattern of bands in the MCD spectrum reports funda-
mentalpropertieslocaltotheheme,suchasspinandoxidation
state,andthisbandpatternwillnotbesignificantlyalteredifthe
spinofthehemeisspin-coupledtoasecondparamagneticspe-
cies. But even a relatively weak interaction of this nature could
preclude the observation of EPR signals. Such an interaction
could, however, be detected via the magnetic field and temper-
aturedependenceoftheMCDintensity.Thesepropertieshave,
therefore,beenstudiedforcd1*-Xusingthe“ratiodata”method
of MCD spectroscopy (RD-MCD). This method was originally
applied to cytochrome bo3to locate and analyze heme o3MCD
bands from the active site FeIIIheme o3-CuB
intense background of low-spin FeIIIheme b bands (44). RD-
MCD allows the immediate identification of transitions in the
MCD spectrum, which arise from paramagnetic species for
whichthespinS?1⁄2.DetailsofthemethodaregiveninRef.44,
andonlyabriefsummarywillbepresentedhere.Forthecaseof
an S ?
tanh(??H?/2 kT), where ? is the Bohr magneton, k is Boltz-
mann constant, H the magnetic field, and T the absolute tem-
perature. ? is a function of the g values, which determines the
magnitude of the Zeeman splitting between the ground state
components, mS? ?1⁄2, at any orientation of the molecule to
the external magnetic field expressed as the spherical polar
angles ? and ?.
IIpair against an
1⁄2 chromophore, the MCD intensity varies as
? ? [gzz
2cos2? ? (gxx
2cos2? ? gyy
2sin2?)sin2?]1/2
(Eq. 3)
Exceptatextremehighmagneticfieldorlowtemperature,out-
side of those used in this work, tanh(??H?/2kT) ? ?H?/2kT,
and so the MCD intensities of S ?1⁄2 systems will be propor-
tional to H/T. Therefore if the MCD spectrum is recorded at
different temperatures, but the magnetic field is adjusted so as
to maintain the H/T ratio, then the contribution to the spec-
trumofS?1⁄2speciesremainsconstant.Variationsinintensity
will be observed only for bands arising from paramagnetic cen-
ters,whicharenotsimpleS?1⁄2species.Fig.7showsRD-MCD
spectra of cd1*-X, from Pp and Ps, recorded at four different
combinations of H/T. It is immediately apparent, for both
enzymes, that the spectra are closely matched in intensity
across the 520–590 nm region. This is entirely consistent with
thepreviousassignmentofthesebandsinFig.6tothelow-spin
FeIIIheme c. In the regions to either side, where heme d1bands
occur,thereareobviousvariationsintheMCDintensities.This
divergence of the spectra is most pronounced in the 590–700
nmregionwherethebandsareexclusivelyfromhemed1.These
RD-MCDdatathereforedemonstratethathemed1incd1*-Xis
part of a paramagnetic but EPR-silent system. The S ?1⁄2 low-
spinFeIIIhemed1mustbeinteractingwithanotherhalf-integer
(Kramers) paramagnet. Heme c is clearly not involved; it
remains EPR detectable and in the RD-MCD spectra gives rise
to bands, which overlie as expected for an isolated S ?1⁄2 spe-
3FeIIIhemes with His/Met ligation give rise to a CT transition near 695 nm.
MCD spectra of the semi-apo form of cd1show that the intensity of this
band in the MCD is two orders of magnitude lower than these heme d1
transitions. G. Kemp and M. R. Cheesman, unpublished.
FIGURE6.LowtemperaturevisibleregionMCDspectraofthecd1*-Xform
of cd1from Pp (a) and Ps (b). Samples were in 1:1 (v:v) 50 mM BTP, D2O, pH*
6.5/glycerol. Spectra were recorded at temperatures of 1.7 K (——), 4.2 K
(– – –), and 10 K (????) and with a magnetic field of 5 tesla.
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cies. Therefore, the species interacting with heme d1can only
be a radical resulting from the reaction between oxidized cd1
and NO2
?.
DISCUSSION
This work has shown that Pp cd1in the oxidized state will
react directly with the physiological substrate NO2
cd1*-X, a complex in which heme d1is paramagnetic but EPR-
silent. This reaction drives a ligand switch at FeIIIheme c from
His/His to His/Met, the conformation associated with enzyme
activity (11–13, 34). Hence, the enzyme has been activated
without passing through the reduced state, something previ-
ously considered essential (6, 11, 34). Dissociation or displace-
mentofTyr25toallowNO2
bedrivenbyreductionofthetwohemes,andthelackofappro-
priate P. pantotrophus cellular components with reduction
potentials low enough to reduce cd1may not be an issue (27).
We have also shown that an equivalent cd1*-X species is
formed in the reaction of NO2
reactivity is not, therefore, unique to enzyme with the ability to
switchhemecligandsbutmaybeapropertyofcytochromescd1
in general. Over twenty years ago, Muhoberac and Wharton
reported that the addition of NO2
cd1results in the loss, from the EPR spectrum, of features asso-
ciated with heme d1(45). The samples, at pH 7.0, were incu-
bated for 24 h before spectra were recorded. There can be little
doubt that this is the same reaction as has been described here
in detail.
MCD showed that the EPR-silent FeIIIheme d1is paramag-
netic but that S ?1⁄2. Because heme c is magnetically isolated,
and the enzyme contains no other cofactors, heme d1must be
interacting with a second paramagnet, presumably a radical
species formed in the reaction with NO2
?to form
?bindingdoesnotthereforehaveto
?with cd1from P. stutzeri. The
?to Pseudomonas aeruginosa
?. The spin interaction
is of sufficient strength to preclude observation of EPR signals
at X-band, and so the magnitude of J, the spin-coupling con-
stant, must therefore be several times that of the microwave
photon energy (?0.3 cm?1). However, the two spins are not
coupled sufficiently strongly to yield a diamagnetic system; the
MCD intensifies significantly between 4.2 and 1.7 K (Fig. 6)
placing an upper limit on J of ?5 cm?1(our preliminary anal-
ysis4of the RD-MCD suggests that J ? 3 cm?1). This order of
magnitude would require that the radical is located in close
proximity to heme d1. For example, in compound I of cyto-
chrome c peroxidase, the exchange coupling between a trypto-
phan radical and an oxoferryl (FeIV?O) heme separated by ?5
Å is ?0.1 cm?1(46, 47).
OnepossibleexplanationisthatasingleturnoverofNO2
taken place (Equation 1) using an electron abstracted from a
nearby residue and so forming a radical. Displacement of the
NO by a second NO2
which low-spin FeIIIheme d1is coupled to the protein radical.
Tyr263is the only fully conserved residue in close proximity to
heme d1and has already been proposed as the location of a
radical species formed during the reaction of reduced cd1with
oxygen (23).
However, although the visible region absorption and MCD
spectraofPpcd1*-Xarecharacteristicoflow-spinFeIIIhemed1,
a band at this specific wavelength (?631 nm, Fig. 1a) has pre-
viouslybeenassignedtoaFeIIIhemed1nitrosylspecies(18,48)
formed in the reaction of NO2
of NO to oxidized cd1from both Pa and Ps also results in
absorptionspectrasimilartothatofPscd1*-X(Fig.1b)(19,49).
Thiswould,atfirst,appeartocontradictourdatabecauseferric
nitrosylhemesarebestdescribedasdiamagneticFeII-NO?spe-
cies (50) and do not give rise to spectra characteristic of FeIII.
Theunpaired?*electronoftheNOislargelytransferredtothe
FeIIIdxz,yzorbitals. But we have previously proposed that the
fully occupied dxz,yzorbitals in the unusual (dxz,yz)4(dxy)1
ground state of low-spin FeIIIheme d1could destabilize this
modeofNObinding(5).Hemed1incd1*-Xisclearlyparamag-
netic and may represent a genuine FeIII-NO species, in which
the NO ligand itself is the radical species interacting with the
spin of FeIIIheme d1. This interpretation would require that a
transient protein radical is formed during the reaction but dis-
sipates rapidly. A relatively weak coupling between a radical
ligand and the spin of the metal ion to which it is bound is
unusual but not without precedent. In horseradish peroxidase
compoundI,thecouplingbetweentheoxoferryl(FeIV?O)iron
andaradicalontheporphyrinligandis?4cm?1(51,52).Inthe
caseoflow-spinFeIIIhemed1,theunpairedelectronofmetalis
located not in dxz,yzorbitals, which readily interact with NO,
but in the dxyorbital (5), which lies in the plane of the isobac-
teriochlorinandispotentiallyorthogonaltotheNO?*orbitals
in a linear Fe-N-O conformation. However, these putative
FeIII-NO species are stable, and so this property of heme d1
alone is not sufficient to dissociate product. It was previously
reported that addition of the chemical reductant ascorbate to
as-prepared Pa cd1resulted in the appearance of the EPR sig-
?has
?would yield a species [R??d1
III-NO2
?] in
?with FeIIheme d1. The addition
4V. S. Oganesyan, M. R. Cheesman, and A. J. Thomson, manuscript in
preparation.
FIGURE7.RD-MCDSpectraofcd1fromPp(a)andPs(b)inthecd1*-Xform.
Samples were in 1:1 (v:v) 50 mM BTP, D2O, pH* 6.5/glycerol. Spectra were
recordedattemperature/magneticfieldcombinationsof1.75K/0.41T(——),
4.2 K/1.0 T (– – –), 10 K/2.37 T (????), 20 K/4.72 T (–??–??–)(a) and 1.88 K/0.38 T
(——), 4.2 K/1.0 T (– – –), 10 K/2.37 T (????), 20 K/4.72 T (–??–??–)(b).
Activationofcd1bySubstrate
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nature of FeII-NO (53). This was attributed to the presence of
NO2
it cannot be excluded that the species responsible was equiva-
lent to cd1*-X and contained FeIII-NO. This would also yield
ferrousnitrosylEPRsignalsuponreduction.EPRspectraofthe
as-prepared enzyme are not shown (53), and where they are
presented elsewhere (17, 32, 39, 45, 54), no integrations are
reported. Consequently it is not possible to say whether or not
thesesamplescontainedEPR-silenthemed1.Iftheydid,thenit
may be that Pa and Ps cytochromes cd1are prone to retaining
product NO. The contrasting behavior of Pp cd1may be linked
to an ability of Tyr25to displace distal heme d1ligands.
NOevolutionisdetectedduringcd1activityassays,whichare
performed in the presence of excess reductant (13, 55). Yet, in
several studies of the reaction of NO2
enzymes from both Pp and Pa, the reaction stopped after a
single turnover (18–21). Evidently the role of reductant in
product release is complex and may well be related to redox-
linked conformational changes at heme c.
The reaction with NO2
the oxidized Pp enzyme can be activated by substrate but at a
ratewhichisseveralordersofmagnitudeslowerthanturnover.
Itisunlikelythereforethatthisspeciesisdirectlyinvolvedinthe
catalyticcycleinthisexactform.However,theinvolvementofa
transient protein radical in NO2
additional redox center and raises the possibility that heme d1
does not access the FeIIstate during turnover of NO2
wouldperhapsexplaintheobservationsthatcarbonmonoxide,
aligandtoFeIIhemes,inhibitsthereductionofO2byPacd1but
not the reduction of NO2
ligand to FeIIIheme, inhibits both activities (56). The idea that
the enzyme turns over too rapidly for reversion to the His/His
hemectooccur(13,26)maystillbevalid.However,itshouldbe
considered that this slow reversion is prevented by substrate
binding rather than re-reduction of heme d1.
In summary, we have shown that cd1in the oxidized state
reacts directly with its substrate NO2
HistoHis/Metligandswitchrequiredforactivity.Thereaction
also occurs with cd1s that do not undergo ligand switching and
do not require activation. The resulting novel species contains
low-spinFeIIIhemed1thatisrenderedEPR-silentbyspin-cou-
pling to a radical that we propose is bound nitric oxide formed
in the reaction.
?intheenzyme,whichwasbeingreducedtoNO.However,
?with the fully reduced
?constitutes a mechanism by which
?reduction would represent an
?. This
?. In contrast cyanide ion, a strong
?driving the heme c His/
Acknowledgments—We thank Dr. James Allen for valuable discussions
andProf.AndrewThomsonforcriticalreadingofthemanuscript.
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