Biochem. J. (2013) 451, 389–394 (Printed in Great Britain)
389
doi:10.1042/BJ20121406
Unexpected weak magnetic exchange coupling between haem and
non-haem iron in the catalytic site of nitric oxide reductase (NorBC) from
Paracoccus denitrificans 1
Jessica H. VAN WONDEREN*, Vasily S. OGANESYAN*, Nicholas J. WATMOUGH†, David J. RICHARDSON†,
Andrew J. THOMSON*2 and Myles R. CHEESMAN*2
*School of Chemistry, University of East Anglia, Norwich Research Park, Norwich NR4 7TJ, U.K., and †School of Biological Sciences, University of East Anglia, Norwich Research Park,
Norwich NR4 7TJ, U.K.
Bacterial NOR (nitric oxide reductase) is a major source of
the powerful greenhouse gas N2 O. NorBC from Paracoccus
denitrificans is a heterodimeric multi-haem transmembrane
complex. The active site, in NorB, comprises high-spin haem b3 in
close proximity with non-haem iron, FeB . In oxidized NorBC, the
active site is EPR-silent owing to exchange coupling between FeIII
haem b3 and FeB III (both S = 5/2). On the basis of resonance Raman
studies [Moënne-Loccoz, Richter, Huang, Wasser, Ghiladi, Karlin
and de Vries (2000) J. Am. Chem. Soc. 122, 9344–9345], it has
been assumed that the coupling is mediated by an oxo-bridge
and subsequent studies have been interpreted on the basis of
this model. In the present study we report a VFVT (variablefield variable-temperature) MCD (magnetic circular dichroism)
study that determines an isotropic value of J = − 1.7 cm − 1
(H = −J S1 · S2 ) for the coupling. This is two orders of magnitude
smaller than that encountered for oxo-bridged diferric systems,
thus ruling out this configuration. Instead, it is proposed that weak
coupling is mediated by a conserved glutamate residue.
INTRODUCTION
A bridging oxygen ligand (H2 O, − OH or O2 − ) was identified
in the Pseudomonas NorBC structure, which also supports
suggestions that a conserved glutamate residue could provide an
additional ligand to FeB [8,13,14]. However, absorption spectra
of the crystals showed that substantial reduction occurs upon
exposure to the X-ray beam [11]: the structure provides an
invaluable framework to which other data can be referenced, but
is not that of fully oxidized NorBC. Spectroscopic studies are
therefore essential to understand how metal cofactor configuration
alters with redox state. FeIII haem b3 in oxidized NorBC from
Paracoccus denitrificans gives rise to an electronic CT (chargetransfer) transition characteristic of high-spin FeIII haem, but at the
unusually short wavelength of 595 nm [6,8]. Resonance Raman
showed that haem b3 is five-co-ordinate: since lack of EPR signals
necessitates distal ligand bridging to FeB III , it was concluded
that the proximal histidine residue is dissociated from haem b3 .
Sensitivity of Raman bands to isotopic oxygen substitution was
taken to indicate that X is a bridging oxygen ligand and oxo was
favoured over hydroxo [7,9,10].
Many di-iron complexes, reported as models for non-haem
iron proteins, have been magnetically characterized. Ligandmediated coupling between high-spin FeIII ions results in a
negative isotropic exchange coupling (J) that is significantly larger
for μ-oxo than for μ-hydroxo (reviewed by Solomon et al. [15]).
All of the J-values discussed in the present study are referenced
to the H = −J S1 · S2 convention. Isotropic J-values of − 154
cm − 1 and − 216 cm − 1 identified oxo-bridged diferrric sites in
oxy-haemrythrin [16] and RNR (ribonucleotide reductase) from
Escherichia coli [17]. In contrast, FeIII –FeIII coupling in MMO
(methane mono-oxygenase) from Methylosinus trichosporium
of − J = 15 cm − 1 indicated μ-hydroxo [18]. As the bridge is
Bacterial denitrification is a fundamental component of the global
nitrogen cycle and involves the reduction of nitrate (NO−3 ) to
dinitrogen (N2 ) in four steps:
NO−3 → NO−2 → NO → N2 O → N2
Use of nitrate fertilizer stimulates soil denitrification and the
emission into the atmosphere of nitrous oxide (N2 O), a greenhouse
gas 300 times more potent than CO2 , with a half-life of >100 years
[1]. N2 O has now become the most serious ozone-depleting
emission [2]. NOR (nitric oxide reductase), a haem-containing
enzyme in the cytoplasmic membrane of denitrifying bacteria [3]
that is responsible for the majority of N2 O production, catalyses
the one-electron reduction of nitric oxide:
2NO + 2H+ +2e− → N2 O + H2 O
(1)
NOR is also used by the opportunistic pathogen Pseudomonas
aeruginosa, in the lung of cystic fibrosis patients, to detoxify
macrophage-produced NO [4]. A suggestion, on the basis of
primary sequence analyses [5], that NOR is structurally related to
HCOs (haem-copper oxidases) was supported by spectroscopic
studies [6–10] and by the crystal structure of NorBC from
Ps. aeruginosa [11]. NorBC contains two low-spin haems
and a haem/non-haem iron active site, arranged within two
protein subunits as illustrated in Figure 1. FeB takes the place
of the copper ion found at the active site of HCOs. As with HCOs,
the active site of oxidized NOR is EPR-silent and it is assumed
that haem b3 and FeB are spin-coupled via a bridging ligand (X in
Figure 1) [6,8,12,13].
Key words: denitrification, haem, magnetic circular dichroism
(MCD), nitric oxide reductase (NOR), non-haem iron.
Abbreviations used: CT, charge-transfer; HCO, haem-copper oxidase; MCD, magnetic circular dichroism; nIR, near IR; NOR, nitric oxide reductase;
RD-MCD, ratio-data MCD; RT-MCD, room temperature MCD; VFVT, variable-field variable-temperature; VT-MCD, variable temperature MCD; ZFS, zero-field
splitting.
1
This paper is dedicated to the memory of Colin Greenwood.
2
Correspondence may be addressed to either of these authors (email [email protected] or [email protected]).
c The Authors Journal compilation c 2013 Biochemical Society
390
J. H. Van Wonderen and others
Figure 1 Subunit arrangement and metal cofactor content of NorBC in the
595 form
All iron atoms are in the FeIII oxidation state.
responsible for mediating the coupling between haem b3 and FeB ,
an accurate determination of J should aid identification of this
ligand in NorBC. Although the haem b3 –FeB interaction has been
described as antiferromagnetic [7,9], the nature of the coupling is
unknown, beyond that it precludes observation of the EPR signals
at the X-band. In principle, the coupling can be determined by
analysing magnetic field- and temperature-dependent variations
in the MCD (magnetic circular dichroism) intensity of the 595 nm
band [19]. Contributions from other forms, and from the other
haems, can be minimized or eliminated by working at this
wavelength. Residual contributions from haems b and c can be
removed entirely by careful choice of measurement conditions
that exploit the fact that S = 1/2 centres have a relatively simple
magnetic field and temperature dependence. In the present paper
we describe how we have used this approach to determine, for
the first time, the magnitude of the spin coupling between FeIII
haem b3 and FeB III in the 595-form of NorBC from Paracoccus
denitrificans.
EXPERIMENTAL
NorBC was purified as described from the P. denitrificans
strain 93.11 [20]. Spectroscopic samples were prepared as
buffered solutions in 2 H2 O [21]. All solutions contained
0.05 % dodecyl-maltoside and 20 mM BTP {Bis-Tris propane;
1,3-bis[tris(hydroxymethyl)methylamino]propane} at pH* = 7.4.
pH* is the apparent pH of the 2 H2 O solutions measured using
a standard glass pH electrode. For low-temperature spectra, an
equivalent volume of glycerol was added as a glassing agent. MCD
spectra were recorded on JASCO circular dichrograph models
J810 and J730 used in conjunction with an Oxford Instruments
SM4 split-coil superconducting solenoid capable of generating
fields of up to 5 T. Intensities of spectra presented are referred
to concentrations on the basis of an extinction coefficient of 300
mM − 1 ·cm − 1 for the Soret absorption band near 410 nm [12].
RESULTS
Electronic absorption
Figure 2 (solid trace) is the absorption spectrum of P. denitrificans
NorBC showing the distinctive 595 nm band. The 300–580 nm
region contains overlapping bands from all three haems. For
high-spin FeIII haems, two CT transitions (CT1 and CT2 ) occur
at wavelengths longer than 580 nm [21]. Both could potentially
yield information on the magnetic properties of haem b3 and
therefore on the nature of the haem b3 –FeB interaction. CT1 , at
800–1300 nm, is weak and rarely detected by absorption, but CT2
is readily observed at 580–660 nm. Preparations of oxidized P.
denitrificans NorBC exhibit an electronic transition at 595 nm
[6,8,22], but we note that this band shows minor variations
c The Authors Journal compilation c 2013 Biochemical Society
Figure 2 UV–visible region electronic absorption spectra of NorBC in the
595 and the 622 forms
Spectra of the 595 form (continuous line) and the 622 form (broken line). These spectra are
plotted using ε = 300 mM − 1 ·cm − 1 for the Soret band at ∼410 nm [12].
in intensity. Preparations with low 595 nm absorption intensity
instead give rise to weaker bands at longer wavelengths [23]
and it needs to be ensured that the presence of these bands
does not compromise analysis of the 595 nm transition. The
broken trace in Figure 2 shows the spectrum of a preparation
containing anomalously low 595 nm intensity, but weak intensity
at ∼600–660 nm. The difference spectrum (Figure 2, inset) shows
the absorption to be significantly red-shifted from the 595 nm
transition and centred at ∼622 nm. This shift in wavelength along
with differences in magnetic properties (discussed below) ensures
that low levels of these other forms will not adversely affect
analyses of the MCD at 595 nm.
VFVT (variable-field variable-temperature) MCD
For a sample of predominantly 595 form NorBC {∼90 % as
judged by RT-MCD (room temperature MCD) [23]}, visibleregion MCD spectra were recorded at 18 temperatures between
1.7 and 220 K, using a field of 5 T, to give the dataset εVT
(Figure 3A). At these temperatures, MCD bands of low-spin FeIII
haems b and c are 1–2 orders of magnitude more intense than
those of high-spin haem b3 and dominate the spectra [21]. The
nIR (near IR) region, Figure 3(B), contains two low-spin haem CT
bands at wavelengths characteristic of the His/His (1550 nm) and
His/Met (1900 nm) ligation of haem b and haem c respectively.
These bands match those reported in the RT-MCD [8]: there is
no evidence for a haem b3 spin-state change at low temperature
as observed for the Pseudomonas stutzeri enzyme [12]. The only
exception to the dominance of low-spin bands occurs near 595 nm,
where the bandshape changes with temperature, suggesting the
presence of transitions from the 595 form active site (expanded
in Figure 3C). The absence of low-temperature MCD bands at
610–630 nm (Figure 3C) suggests that, in the 622 form, haem b3
and FeB are strongly coupled to yield a diamagnetic active site
and will not therefore compromise analysis of the 595 nm bands.
To extract active site magnetic parameters, the challenge is one
of analysing weak high-spin haem MCD against a background of
intense low-spin transitions. This can be achieved using RDMCD (ratio-data MCD) [19]. The magnetic interaction between
the two high-spin FeIII ions can be described by an effective
spin-Hamiltonian involving an exchange coupling tensor J:
b3
H = gβ H Ŝ + Db3 Ŝ2z − Ŝ2 /3 + E b3 Ŝ2x − Ŝ2y
FeB
+ gβ H Ŝ + DFeB Ŝ2z − Ŝ2 /3 + E FeB Ŝ2x − Ŝ2y
−Ŝb3 · J · SFeB
(2)
Active site structure of nitric oxide reductase
391
Figure 4 Energy level schemes for the two S = 5/2 spins of haem b 3 and
FeB in weak and strong coupling models
The rhombic ZFS parameters used were ( E FeB / D FeB ) = 1/3 and ( E b3 / D b3 ) = 0.
Figure 3
VFVT MCD spectra of P. denitrificans NorBC
(A) VT-MCD spectra in the visible region. Spectra were recorded using a magnetic field of 5 T
and temperatures of 1.73, 2.25, 2.95, 3.86, 4.22, 6.10, 7.90, 10.0, 15.0, 20.0, 29.6, 44.6, 64.5,
85.0, 110, 151, 182 and 220 K. All spectroscopic features increase in intensity with decreasing
temperature. (B) VT-MCD spectra in the nIR region. Spectra were recorded using a magnetic
field of 5 T and temperatures of 1.73, 4.22, 10.0 and 50.0 K. (C) Expansion of the 595 nm
region of (A). (D) RD-MCD spectra in the visible region. Spectra were recorded using magnetic
fields and temperature combinations (in Tesla/Kelvin) of 0.247/1.73, 0.606/4.24, 1.714/12.0,
2.857/20.0, 3.857/27.0 and 5.000/35.0. The bi-signate feature at 595 nm increases in intensity
with decreasing temperatures. (E) Difference spectra of the 595 nm MCD band obtained from
the spectra in (D). (F) RT-MCD spectra in the 595 nm region of NorBC (dotted line) (×1/3);
hydroxide-bound FeIII -protoporphyrin IX (dashed line); and water-bound FeIII -protoporphyrin
IX (continuous line). The mid-point of each bi-signate band is shown by arrows at 595, 606 and
616 nm respectively.
where []b3 and []FeB are local spin-Hamiltonians for haem b3 and
FeB , each containing a Zeeman term, plus axial (D) and rhombic
(E) ZFS (zero-field splitting) parameters. Figure 4 shows energy
level schemes in two limiting regimes. Strong exchange coupling
(|J||D|) yields a diamagnetic ground state. High-spin haem ZFS
parameters are substantially larger than those of non-haem iron:
under weak exchange (|J|≈|D|), each of the three well-separated
haem b3 doublets couples with six FeB levels to give three groups
of energy levels each containing twelve states. The electronic
transitions of FeIII haems are xy-polarized [24]. Consequently the
MCD temperature and field dependence is wavelength invariant
and the intensity can be described as a superposition of the
following contributions:
ε (λ, T, H ) = Cls (λ) Sz (T, H )ls + Chs (λ) Sz (T, H )hs
+A (λ)
βH
3
(3)
where ε(λ,T,H) is total MCD intensity at wavelength λ,
temperature T and field H; terms in Cls (λ) and Chs (λ)
represent temperature-dependent parts due to the two low-spin
haems and to high-spin haem b3 respectively; A(λ) represents the
temperature-independent contributions from all three. Exchange
coupling will not significantly perturb the form of haem b3 MCD
bands. However, changes to ground state magnetic properties
resulting from an interaction with FeB will be reflected in the
temperature- and field-dependent factor Ŝz (T, H )hs . This can
be calculated by diagonalization of the Hamiltonian matrix
constructed from eqn (2). Expectation values of the spin operator
Ŝbz 3 for all resulting states can be calculated and averaged both
thermally and orientationally [19], but first contributions from the
terms in A(λ) and Cls (λ) must be removed to obtain Chs (λ). The
temperature- and field-dependent factor (Sz (T, H ))ls for either
low-spin haem can be calculated from:
1
Sz (T, H ) =
4π
π
ls
0
0
2π
cos2 θ sinθ
βH
gzz
tanh −
dθ dφ
2kT
(4)
where angles θ and φ define the orientation of an
individual
haem relative to the applied field, and =
2
2
2 cos2 φ + g 2 sin2 φ . RD-MCD exploits the fact
gzz cos θ + gxx
yy
that the dependence of the MCD intensity on T and H has this
relatively simple form for an S = 1/2 centre. Recording spectra at
combinations of field and temperature that maintain a fixed H/T
ratio results in a constant contribution to the paramagnetic term
Cls (λ) from low-spin haems: variations in MCD intensity then
arise only from haem with a spin state other than S = 1/2.
RD-MCD spectra were recorded at six combinations of H and
T with a fixed ratio of 0.143 T·K − 1 yielding a second dataset,
εRD (Figure 3D). At most wavelengths, the scans virtually
overlie because the spectra are dominated by S = 1/2 low-spin
haems. However, at certain wavelengths, especially in the region
of the 595 nm band, spectra show RD-MCD intensity variation,
demonstrating the presence of a paramagnetic chromophore with
S=1/2. A measure of A(λ), the temperature-independent contribution from all haems was obtained by extrapolating the VT-MCD
(variable temperature MCD) intensity (εVT ) to high temperature
c The Authors Journal compilation c 2013 Biochemical Society
392
J. H. Van Wonderen and others
more sensitive to Db3 than is ε RD . Figure 5(B) shows a plot
of the peak-to-trough (585–593 nm) MCD intensities taken from
the ε VT dataset. These data contain the temperature-dependent
part of the VT-MCD of all three haems as described by the two
terms Cls and Chs of eqn (3). The invariant part of the RD-MCD
in ε RD was used to fix low-spin contributions to ε
VT [19]: this
is shown as the dotted trace in Figure 5(B). The dashed line shows
the simulated contribution from haem b3 using the Hamiltonian
of eqn 2. The excellent fit of the solid line, showing the
addition of these two simulated contributions, to the experimental
data supports the haem b3 ZFS parameters used in the simulations
of Figure 5(A).
DISCUSSION
Figure 5
RD-MCD and VT-MCD plots of the HS FeIII haem b 3 CT2
(A) RD-MCD plot of the HS FeIII haem b 3 CT2 (595 nm) band intensity extracted from the ε
RT
MCD dataset for NorBC. 䊉, extracted data points; continuous line, simulation as described
in text using D b3 = 5 cm − 1 , ( E b3 / D b3 ) = 0.06, D FeB = 1.0 cm − 1 , ( E FeB / D FeB ) = 1/3,
J = − 1.7 cm − 1 . (B) VT-MCD plot of the HS FeIII haem b 3 CT2 (595 nm) band intensity
extracted from the ε VT MCD dataset for the 595 form of NorBC: 䊊, extracted data points;
dotted line, simulated LS FeIII contributions; dashed line, simulated contribution from coupled
active site using D b3 = 5 cm − 1 , ( E b3 / D b3 ) = 0.06, D FeB = 1.0 cm − 1 , ( E FeB / D FeB ) = 1/3,
J = − 1.7 cm − 1 ; continuous line, sum of these two contributions.
(1/T→0). This was subtracted from each εVT spectrum to
give a corrected dataset ε
VT , containing the paramagnetic
intensity of the three haems. A (λ) was similarly subtracted from
the εRD , after scaling appropriately for the magnetic fields
used, giving ε
RD . The fixed contribution from low-spin haems
could then be removed by subtracting one ε
RD spectrum from
each of the others, resulting in the difference spectra shown in
Figure 3(E). Peak-to-trough (585–593 nm) intensities of these
difference spectra have been normalized and the results plotted
against 1/T (Figure 5A). These data are dependent solely on
the factor Sz (T, H )hs and therefore carry active site magnetic
parameters. The continuous line is the optimum simulation
achieved using the spin Hamiltonian of eqn (2). For mononuclear
non-haem iron proteins and model complexes, D is typically
2 cm − 1 [25–27]. Our simulations are insensitive to variations
in DFeB within this range and so this was set to 1 cm − 1 .
Similarly, changes in (E FeB /DFeB ) produced negligible effects
and this was fixed at 1/3, consistent with the low-symmetry
observed for other FeIII non-haem iron species. Simulations were
then performed by systematically varying Db3 , (E b3 /Db3 ) and
J. The fit of Figure 5(A) was achieved using Db3 = 5 cm − 1 ,
(E b3 /Db3 ) = 0.06 and an isotropic coupling constant of J =
− 1.7 cm − 1 .
For RD-MCD measurements, at the lowest temperature used
(∼1.7 K), the field was set sufficiently high to obtain acceptable
signal intensity. With the fixed H/T ratio, the solenoid maximum
current then defined 35 K as the highest temperature at which
data could be collected. The VT-MCD provides a second route
to parameters describing the active site, but one yielding a larger
dataset, ε
VT , measured over a wider temperature range. As
found in a previous study of cytochrome bo3 [19], this dataset is
c The Authors Journal compilation c 2013 Biochemical Society
The present study has shown that the EPR-silent 595 form active
site is not diamagnetic due to strong coupling, but is paramagnetic
down to 1.7 K. A measurement of the coupling has been made for
the first time, resulting in an isotropic J-value of − 1.7 cm − 1 .
In a study of 34 oxygen-bridged (O2 − , HO − and RO − ) diferric
complexes, Gorun and Lippard [28] showed that J is relatively
insensitive to the M-O-M angle (φ o ), but determined primarily
by the length of the exchange pathway. Weihe and Güdel [29]
further developed this approach for oxo-bridged complexes. They
modelled the dependence of J on the average Fe–O bond length
(rav ) and φ o , confirming that the latter is of minor importance:
− JWG = 1.337 × 108 3.536 + 2.488cosφo + cos 2 φo
ex p (−7.909rav )
(5)
The experimental J-values for the 32 complexes lay
within 15 % of those predicted by eqn (5). This is also
true for the complexes reported subsequently (Supplementary
Table S1 at http://www.biochemj.org/bj/451/bj4510389add.htm).
Furthermore, if the Weihe–Güdel [29] formula is applied to
structurally and magnetically characterized oxo-bridged di-haem
or haem/non-haem iron complexes, it predicts J as successfully
as it does for non-haem iron complexes (Supplementary Table
S1). Thus it can reasonably be assumed that − J for oxo-bridged
haem/non-haem iron structures should also fall in the range
110–270 cm − 1 [29,30]. Our experimentally determined value of
− J = 1.7 cm − 1 is two orders of magnitude smaller and rules out
an oxo-bridge in the 595 form active site.
In the Pseudomonas NorBC structure, haem b3 is bound by a
proximal histidine and bridged to FeB by a single oxygen. The Fe–
O bond lengths are 1.9 and 2.0 Å (1 Å = 0.1 nm) [11], significantly
longer than the 1.73–1.82 Å found for oxo-bridges, but consistent
with hydroxide bridging. However, absorption spectra showed
that reduction of the low-spin haems occurs rapidly upon exposure
to X-rays [11]. The structure is therefore consistent with our
previous study showing that, when haems b and c are reduced,
the distal haem b3 ligand becomes hydroxide and the proximal
histidine residue rebinds [8], but it provides no evidence for the
presence of hydroxide bridging in the 595 form.
Hay et al. [31] suggested that decreasing the charge on the
bridging oxygen lengthens the Fe–O bonds and diminishes
coupling. Thus − J should be smaller for RO − bridges than oxobridges: this is borne out by observation. Haase and co-workers
analysed 34 phenoxo-, alkoxo- and hydroxo-bridged non-haem
diferric complexes [30], for which − J lies in the range of 10–60
cm − 1 . Again, the magnitude of J was found to depend upon the
coupling pathway and the influence of φ o is negligible:
− JH = 2 × 107 ex p (−6.8rav )
(6)
Active site structure of nitric oxide reductase
Figure 6 Proposed structure for the diferric active site of the 595 form of
NorBC incorporating a carboxylate bridge
For hydroxo-bridging, − J falls in the narrower range of
10–35 cm − 1 , again significantly larger than 1.7 cm − 1 . There
are few characterized hydroxo-bridged haem complexes and so
generalizations from non-haem iron should be applied with care.
The reported − J values of >50 cm − 1 [32] and 9 cm − 1 [33]
suggest that coupling may be weaker for some haem complexes,
but still larger than − J = 1.7 cm − 1 , making μ-hydroxide also
very unlikely in the 595 form. A further decrease in coupling is
anticipated on changing from hydroxide to water [31]. Considered
in isolation, a − J of 1.7 cm − 1 makes water a plausible bridge.
However, the position of the CT2 band for five-co-ordinate waterligated FeIII –protoporphyrin IX is significantly red-shifted from
595 nm (Figure 3F) as it is for analogous haem structures in
proteins (e.g. [34]).
For the 595 form, magnetic coupling and the CT2 wavelength
rule out bridging by simple oxygen ligands, H2 O, HO − or O2 − .
The confines of the active site and limited availability of potential
ligands severely restricts candidates for the species that mediates
coupling. We suggest that this role is fulfilled by the glutamate
residue observed as an FeB ligand in the structure of partially
reduced Pseudomonas NorBC [11] (Figure 6). Carboxylate
bridges, observed in reduced non-haem iron proteins in a μ-1,3
conformation, mediate couplings of − J = 1–5 cm − 1 [35–38].
The results of the present study rule out oxo-bridging in the 595
form, but some rationale is required for resonance Raman studies
that originated this model. Both studies reported two prominent
bands at 760–880 cm − 1 and suggested these are due to ν as (Fe-OFe) modes of different conformations of the oxo-bridge [9,10]. In
H2 18 O, one mode, at 811–813 cm − 1 , shifted to lower energy by
∼30 cm − 1 , consistent with exchange of O2 − , but it was not clear
why the second mode (833 cm − 1 ) was largely unaffected. If a
μ-oxo ligand is ruled out, the H2 18 O-sensitive mode could
be due to the hydroxide-bridged 622 form, but the origin
of the second mode remains unclear. A ligand that would
not exchange, but could give rise to bands at these energies,
is peroxide. This ligand, bound in various conformations to
mononuclear or dinuclear FeIII , gives rise to an asymmetric
O–O stretch, ν as , in the range of 790–932 cm − 1 [39–42].
Although peroxide ion exists as a bridge in several dinuclear
non-haem iron enzyme intermediates, it mediates strong antiferromagnetic coupling (e.g. [43–47]), and if present in
595 form NorBC is more likely to be a ligand to FeB . Thus,
in the 595-form, weak coupling could be provided by bridging
glutamate with peroxide as an η2 ligand to FeB (Figure 6).
In summary, we have measured an unexpectedly small value
of − J = 1.7 cm − 1 for the magnetic exchange between haem
b3 and FeB in 595 form NorBC. This overturns the accepted
oxo-bridged model, but is consistent with carboxylate-mediated
393
coupling. A bridging conformation contrasts with the structure of
partially reduced Pseudomonas NorBC in which the glutamate is
bound only to FeB [11], suggesting a redox-linked conformational
flexibility. The mechanisms proposed for NOR involve the
reoxidation, by 2NO, of a diferrous active site, but are generally
divided into two categories: trans, whereby a nitric oxide molecule
binds to each of haem b3 and FeB ; and cis, in which both substrate
molecules bind to FeB [7,9,10,48]. Each category could include
a number of mechanisms differing in the order of entry into
the protein of the electrons, protons and substrate molecules.
Dissociation from haem b3 of the glutamate residue following
reduction of haems b and c constitutes a mechanism whereby NO
binding to haem is blocked until electrons are present in the protein
and so may have a bearing on the order of events. Furthermore
this glutamate has been implicated in a proton-transfer pathway
from the periplasmic surface to the active site [14], a role it
would not fulfil while in a bridging conformation: electron uptake
could therefore gate proton uptake. Cyanide ion (isoelectronic
with NO + ) binds more strongly, by three orders of magnitude, to
partially reduced NorBC than to the 595 form [49], implying that
the active site is inaccessible to substrate prior to electron uptake.
AUTHOR CONTRIBUTION
Jessica Van Wonderen prepared samples, recorded spectroscopic
data and contributed to interpretation of data. Vasily Oganesyan
performed simulations and analysis of magnetization data. Myles
Cheesman and Andrew Thomson planned the experiments and
interpreted the data. Myles Cheesman wrote the paper. Nicholas
Watmough and David Richardson provided enzyme samples.
Nicholas Watmough, Myles Cheesman, David Richardson and
Andrew Thomson secured funding for the project. All authors
discussed the results and commented on the paper.
ACKNOWLEDGEMENTS
M.R.C. thanks Professor Walter Zumft for helpful suggestions
during the preparation of this paper.
FUNDING
This work was supported by the Biotechnology and Biological
Sciences Research Council grants [grant numbers BBC0077191,
B18695 and BBE0132521] and studentship [grant number
02A1B08117] and a Wellcome Trust award from the Joint Infrastructure Fund for Equipment.
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Biochem. J. (2013) 451, 389–394 (Printed in Great Britain)
doi:10.1042/BJ20121406
SUPPLEMENTARY ONLINE DATA
Unexpected weak magnetic exchange coupling between haem and
non-haem iron in the catalytic site of nitric oxide reductase (NorBC) from
Paracoccus denitrificans 1
Jessica H. VAN WONDEREN*, Vasily S. OGANESYAN*, Nicholas J. WATMOUGH†, David J. RICHARDSON†,
Andrew J. THOMSON*2 and Myles R. CHEESMAN*2
*School of Chemistry, University of East Anglia, Norwich Research Park, Norwich NR4 7TJ, U.K., and †School of Biological Sciences, University of East Anglia, Norwich Research Park,
Norwich NR4 7TJ, U.K.
Table S1
units*
Predicted and observed values of the isotropic exchange coupling constant, J , for example complexes containing μ-oxo- and μ-hydroxo-diferric
Complex
μ-Oxo and μ-hydroxo dinuclear non-haem iron†
[L1 (H2 O)Fe(μ-O)Fe(OH2 )L1 ](ClO4 )4 ·2H2 O]‡
[FeIII (bpc)(H2 O)]2 O§
{[Fe(pca)3 ]2 O}2 − Fe2 O(O2 CCH3 )2 (Tp)2
Fe2 O(O2 CCH2 OCH3 )2 (Tp)2
[Fe2 O(O2 CCH2 N(CH3 )3 )2 (Tp)2 ]2 +
Fe2 O(O2 CCF3 )2 (Tp)2
{[(salten)Fe]2 (OH)}4 + ¶
[Fe2 (OH)(O2 CCH3 )2 (Tp)2 ] +
[Fe2 (OH)(O2 CCH2 Cl)2 (Tp)2 ] +
μ-Oxo and μ-hydroxo di-haem
μ-Oxo-[tetraphenylporphine-FeIII ]2
μ-Oxo-[dimethyl-octaethylporphine-FeIII ]2
μ-Oxo-[meso-tetrakis(p -tolyl-porphine)-FeIII ]2
Ethane-linked μ-oxo-[octaethylporphine-FeIII ]2
Ethane-linked μ-hydroxo-[octaethylporphine-FeIII ]2
μ-Hydroxo-[octaethylporphine-FeIII ]2
μ-Oxo mixed haem/non-haem iron
[(5 L)FeIII -O-FeIII (Cl)] + **
[(6 L)FeIII -O-FeIII (Cl)] + ‡‡
[(F8 -TPP)FeIII -O-FeIII (TMPA)(Cl)] + §§
r av (Å)
φ (◦ )
− J WG (cm − 1 )
1.792
1.779
1.778
1.786
1.79
1.78
1.796
1.997
1.956
1.941
180
180
161.6
124
126
126.6
130.9
159
123
124
192
212
216
242
230
248
212
1.759
1.752
1.740
1.774
1.916
1.938
176.1
178.6
178.2
147.9
142.5
146.2
249
263
289
231
1.777
1.790
1.771
157.3
166.7
156.8
220
196
232
− J H (cm − 1 )
− J obs (cm − 1 )
25
34
37
223 [1]
216 [2]
221 [3]
234 [4]
236 [4]
248 [4]
230 [4]
42 [5]
34 [4]
38 [4]
43.9
38
258 [6,7]
250 [8]
284 [9]
253 [10]
9 [11]
>50 [12]
>100 [13] ††
230
216 [13,14]
*Predicted values of − J calculated from reported values of rav (average M–O bond length) and φ o (M-O-M angle) using eqn (5) [15] and eqn (6) [16] of the main text.
†Calculated for complexes reported subsequently to the studies by Weihe and Gudel [15] and by Werner et al. [16].
‡L1 , N,N -bis(1-methylimidazolyl-2-methyl)-N,N -bismethyl-1,2-ethanediamine.
§H2 bpc, 4,5-dichloro-1,2-bis-(pyridine-2-carboxamido)benzene.
pca − , 2-pyrazinecarboxylate.
¶H2 salten, 4-azaheptane-1,7-bis(salicylideneiminate).
**5 is the dianion of 5-(o -O -[(N ,N -bis(2-pyridylmethyl)-2-(5-methoxyl)pyridinemethanamine)phenyl]-10,15,20-tris(2,6-difluorophenyl)porphine.
††Strong antiferromagnetic coupling reported on the basis of room temperature magnetic moment of 3.4 μB .
‡‡6 L is the dianion of 5-(o -O -[(N ,N -bis(2-pyridylmethyl)-2-(6-methoxyl)pyridinemethanamine)phenyl]-10,15,20-tris(2,6-difluorophenyl)porphine.
§§F8 -TPP, [tetrakis(2,6-difluorophenyl)porphyrinate]2 − ; TMPA, tris(2-pyridylmethyl)amine.
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This paper is dedicated to the memory of Colin Greenwood.
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