Controlling a burn: Outer-sphere gating of hydroxylamine oxidation by a distal base in cytochrome P460

Article abstract of DOI:10.1039/c9sc00195f

Ammonia oxidizing bacteria (AOB) use the cytotoxic, energetic molecule hydroxylamine (NH₂OH) as a source of reducing equivalents for cellular respiration. Despite disproportionation or violent decomposition being typical outcomes of reactions of NH₂OH with iron, AOB and anammox heme P460 proteins including cytochrome (cyt) P460 and hydroxylamine oxidoreductase (HAO) effect controlled, stepwise oxidation of NH₂OH to nitric oxide (NO). Curiously, a recently characterized cyt P460 variant from the AOB Nitrosomonas sp. AL212 is able to form all intermediates of cyt P460 catalysis, but is nevertheless incompetent for NH₂OH oxidation. We now show via site-directed mutagenesis, activity assays, spectroscopy, and structural biology that this lack of activity is attributable to the absence of a critical basic glutamate residue in the distal pocket above the heme P460 cofactor. This substitution is the only distinguishing characteristic of a protein that is otherwise effectively structurally and spectroscopically identical to an active variant. This highlights and reinforces a fundamental principal of metalloenzymology: metallocofactor inner-sphere geometric and electronic structures are in many cases insufficient for imbuing reactivity; a precisely defined outer coordination sphere contributed by the polypeptide matrix can be the key differentiator between a metalloenzyme and an unreactive metalloprotein.

Full text of DOI:10.1039/c9sc00195f

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Controlling a burn: outer-sphere gating of
hydroxylamine oxidation by a distal base in
Cite this: DOI: 10.1039/c9sc00195f
cytochrome P460†
All publication charges for this article
have been paid for by the Royal Society
of Chemistry
Meghan A. Smith,
Sean H. Majer,
Avery C. Vilbert and Kyle M. Lancaster
*
Ammonia oxidizing bacteria (AOB) use the cytotoxic, energetic molecule hydroxylamine (NH
2
OH) as
a
source of reducing equivalents for cellular respiration. Despite disproportionation or violent
decomposition being typical outcomes of reactions of NH OH with iron, AOB and anammox heme P460
proteins including cytochrome (cyt) P460 and hydroxylamine oxidoreductase (HAO) effect controlled,
stepwise oxidation of NH OH to nitric oxide (NO). Curiously, a recently characterized cyt P460 variant
from the AOB Nitrosomonas sp. AL212 is able to form all intermediates of cyt P460 catalysis, but is
nevertheless incompetent for NH OH oxidation. We now show via site-directed mutagenesis, activity
2
2
2
assays, spectroscopy, and structural biology that this lack of activity is attributable to the absence of
a critical basic glutamate residue in the distal pocket above the heme P460 cofactor. This substitution is
the only distinguishing characteristic of
a protein that is otherwise effectively structurally and
spectroscopically identical to an active variant. This highlights and reinforces a fundamental principal of
metalloenzymology: metallocofactor inner-sphere geometric and electronic structures are in many
cases insufficient for imbuing reactivity; a precisely defined outer coordination sphere contributed by the
Received 13th January 2019
Accepted 14th February 2019
DOI: 10.1039/c9sc00195f
polypeptide matrix can be the key differentiator between
a metalloenzyme and an unreactive
rsc.li/chemical-science
metalloprotein.
has evolved Fe-based enzymes bearing heme P460 cofactors that
Introduction
12,13
effect the controlled release of electrons from NH
2
OH.
This
Nature has mastered selective redox chemistry using earth- is remarkable given that more commonly reported reaction
abundant rst-row transition metals, affording a wealth of outcomes between NH OH and Fe comprise disproportionation
inspiration toward human leverage of similar chemistry for or uncontrolled production of ill-dened mixtures bearing
2
1
–3
14–17
a sustainable chemical future. Nature's redox enzymes, be a variety of gaseous oxynitrogen species.
Consequently, P460
they metalloenzymes or otherwise, make critical use of well- cofactors represent a valuable system from which to glean
designed outer coordination spheres in order to tune proper- insights into controlled redox chemistry.
4
–7
ties such as reduction potentials. They also make use of these
motifs to impart substrate chemo- and regioselectivity as from aerobic ammonia oxidizing (AOB), anaerobic ammonia
P460 cofactors are modied c-type hemes found in enzymes
8,9
exemplied by the cytochromes P450. This tuning is crucial oxidizing (anammox), methanotrophic, and—possibly—deni-
for successful biological energy transduction, where metabolic trifying bacteria.
18,19
P460 hemes are distinguished from
enzymes such as those in the Krebs cycle must effect controlled canonical c-hemes by additional covalent porphyrin-amino acid
release of energy from chemical fuel via product selectivity. cross-links. In octaheme enzymes, such as hydroxylamine
Nitrication—a nitrogen-based energy transduction pathway in oxidoreductase (HAO), two cross-links are contributed by
which ammonia (NH
arena in which to study this phenomenon. A key step of nitri- disposed C
3
) serves as the input fuel—affords a fresh a tyrosine, which binds through its phenoxy O and its ortho-
to the porphyrin meso- and a-pyrrolic carbons,
cation is the 3-electron oxidation of cytotoxic and energetic respectively (Fig. 1a). In monoheme cytochrome (cyt) P460
3

10,11
hydroxylamine (NH OH) to form nitric oxide (NO).
Nature enzymes, a lysine sidechain forms a single N–C bond to
a macrocyclic meso carbon (Fig. 1b). The cofactor macrocycle
2
12,20
remains dibasic in either case,
although the cross-link and
Department of Chemistry and Chemical Biology, Baker Laboratory, Cornell University, macrocycle ruffling together afford cofactors with signicantly
Ithaca, NY 14853, USA. E-mail: kml236@cornell.edu
red-shied UV-vis absorption proles whose absorptivities are
†
Electronic supplementary information (ESI) available: Experimental methods,
representative trials for and reduction potential determination, UV-vis
absorption comparison gure, structural comparison gures. See DOI:
0.1039/c9sc00195f
20,21
diminished relative to standard c-hemes.
To date, heme
K
d
P460 cofactors have been shown to effect selective oxidation of
NH
2
OH to NO through formation of a sequence of Fe-nitrosyl
1
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Fig. 1 Heme P460 cofactors found in N. europaea (a) hydroxylamine Fig. 3 Cyt P460 from N. europaea (a, PDB ID: 2JE3) and Nitrosomonas
oxidoreductase (PDBID: 4FAS) and (b) cytochrome P460 (PDBID: sp. AL212 (b, PDB ID: 6AMG). The presence of a distal carboxylate
2JE3). Adapted from ref. 22.
(Glu97) in the N. europaea variant marks a key difference from the
NH OH oxidation-incompetent AL212 variant, which has an alanine
Ala131) occupying the position.
2
(
intermediates (Fig. 2). In HAO catalysis, the NO product rapidly
ꢀ
10
dissociates and is ultimately oxidized by AOB to nitrite (NO ).
In monoheme cyt P460 enzymes, the NO product remains
bound to heme P460 for a sufficient duration to make possible
2
characterization of an array of new variants of both N. europaea
and AL212 cyt P460, we now show that this glutamate residue is
III
2
absolutely required for oxidation of heme P460 Fe –NH OH
nucleophilic attack by a second equivalent of NH OH to form
2
adducts.
22
2 2
N O. The establishment of selective oxidation of NH OH to NO
by hemes P460 overturns decades of convention in which these
cofactors were incorrectly implicated as the enzymatic source of
Results and discussion
Comparison of WT N. europaea and AL212 cyt P460s
ꢀ
10
NO
2
produced by AOB.
˚
We recently reported the 1.45 A crystal structure of a cyt P460
20
WT AL212 cyt P460, although incompetent for turnover of
variant from Nitrosomonas sp. AL212. We noted that, despite
its cofactor exhibiting identical inner-sphere structural features
as well as spectroscopic features to that of the N. europaea
variant used in our prior mechanistic studies, the AL212 cyt
III
6
NH
species that were previously identied as pathway intermedi-
ates in the N. europaea cyt P460 catalytic cycle for NH OH
oxidation to NO/N O (Fig. 2). We explored whether any signi-
2 2
OH, supports stable heme P460 Fe –NH OH and {FeNO}
2
P460 was not competent for NH
2
OH oxidation. Now expanding
2
cant differences in NH OH or NO binding affinities distinguish
catalytically inactive cyt P460s. Representative titration experi-
ments are plotted in Fig. S2,† and aggregate binding affinities
our perspective to include the outer coordination sphere
surrounding the AL212 heme P460 cofactor, we have found that
a key difference from the N. europaea variant is the substitution
of an alanine residue for a glutamate at position 131 (position
2
d 2
are compiled in Table 1. The 298 K K for NH OH binding to
III
Fe cyt P460 at pH 8.0 is 9 ꢁ 1 mM for the WT N. europaea
9
7 in N. europaea, Fig. 3). Carboxylic acid residues have been
24,25
variant. The WT AL212 variant exhibits a modest, 2-fold dimi-
shown to greatly impact the activity of heme proteins.
nution in K
d
to 18 ꢁ 1 mM. Meanwhile, binding affinities for NO
Further, we hypothesized that this glutamate could operate as
III
7
at pH 8.0 are effectively identical: for N. europaea this K is 10 ꢁ
a proton relay during oxidation of Fe –NH
2
OH to {FeNO} —
d
2
mM, while for AL212 it is 8 ꢁ 1 mM. Similarities between the
a reaction that necessarily involves proton transfer. Through
II/III
proteins extend to Fe
reduction potentials, which we deter-
II/III
mined using potentiometric titrations. The Fe
reduction
potential for N. europaea cyt P460 is ꢀ400 ꢁ 5 mV vs. NHE, while
it is ꢀ424 ꢁ 7 mV for AL212 (Fig. S2† and Table 1). UV-vis
absorption spectra and EPR parameters are, likewise, effec-
tively identical between the two variants (Fig. S3† and Table 1).
Overall, these similarities are not surprising given that the
overall cofactor structures in the two cyt P460 variants are
˚
superimposable (RMSD ¼ 0.074 A, Fig. S5†).
Introduction of an outer-sphere glutamate imbues NH OH
2
oxidase activity to AL212 cyt P460
Fig. 2 Working mechanism for NH
2
OH oxidation by heme P460
Despite inner-sphere structural, spectroscopic, and thermody-
cofactors. The steps indicated in black distinguish cyt P460, in which
namic identity between the two cyt P460 cofactors and their
6
the {FeNO} persists and can undergo nucleophilic attack by NH
2
OH.
III
6
adducts, the WT AL212 cyt P460 Fe –NH OH adduct is redox
2
In HAO, no {FeNO} has been observed, presumably due to facile
inactive using either 2,6-dichlorophenolindophenol (DCPIP,
+217 mV), phenazine methosulfate (PMS, +80 mV), or
release of NO, its stoichiometric product of NH
Adapted from ref. 23.
2
OH oxidation.
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Table 1 Spectroscopic and thermodynamic properties of cyt P460 variants under discussion
III
III
Resting Fe
Resting Fe component
III
component 1 (S ¼ 5/2)
2 (S ¼ 5/2)
Fe –NH
2
OH (S ¼ 1/2)
Reduction Potential
(mV vs. NHE)
NH
2
OH
NO
(mM)
Variant
K
d
(mM)
K
d
geff values
E/D
geff values
E/D
g values
WT N. europaea
WT N. sp. AL212
AL212 Ala131Glu
AL212 Ala131Gln
AL212 Ala131Leu
AL212 Ala131Asp
ꢀ400 ꢁ 5
ꢀ424 ꢁ 7
ꢀ428 ꢁ 2
ꢀ406 ꢁ 2
ꢀ381 ꢁ 10
ꢀ388 ꢁ 7
9 ꢁ 1
18 ꢁ 1
16 ꢁ 5
15 ꢁ 3
12 ꢁ 3
19 ꢁ 7
10 ꢁ 2
8 ꢁ 1
5 ꢁ 1
6 ꢁ 1
2 ꢁ 1
4 ꢁ 1
6.57, 5.09, 1.97
6.39, 5.13, 1.97
6.40, 5.14, 1.97
6.51, 5.12, 1.97
6.40, 5.11, 1.98
6.40, 5.12, 1.97
0.03
0.03
0.03
0.03
0.03
0.03
N/A
N/A
2.75, 2.28, 1.54
2.84, 2.25, 1.44
2.86, 2.27, 1.46
2.78, 2.28, 1.49
2.80, 2.27, 1.46
2.86, 2.25, 1.44
6.00, 5.52, 1.99
6.00, 5.51, 1.99
6.03, 5.53, 1.99
6.00, 5.48, 1.99
6.03, 5.50, 1.99
0.012
0.012
0.012
0.012
0.012
6
7
hexaammonium ruthenium(III) chloride ([Ru(NH
mV) as oxidants. When 10 mM NH OH and 70 mM DCPIP were ground consumption of DCPIP—monitored by following its
added to WT AL212 cyt P460, no cyt P460 intermediates (i.e. absorbance at 605 nm—occurs, which can also be observed in
3
)
6
]Cl
3
, ꢀ8.3 {FeNO}, {FeNO} ) were observed (Fig. 4a). Only basal, back-
2
ꢀ
1
the absence of any cyt P460: 0.44 ꢁ 0.19 mM DCPIP$mM
ꢀ
1
NH
2
OH$min . By contrast, with 1 mM WT AL212 cyt P460 this
process occurs with a specic activity of 0.43 ꢁ 0.02 mM
ꢀ
1
ꢀ1
DCPIP$mM NH
2
OH$min (Fig. 5).
Introduction of glutamate to position 131 in the AL212 cyt
III
P460 imbued catalytic competence for Fe –NH
2
OH oxidation
to NO and subsequent generation of N O by the AL212 protein.
2
Again using 10 mM NH OH and 70 mM DCPIP, the Ala131Glu
2
6
AL212 variant exhibits rapid formation of the cyt P460 {FeNO}
species concomitant with rapid oxidant consumption (Fig. 4b).
Using DCPIP as oxidant, Ala131Glu AL212 cyt P460 oxidizes
ꢀ1
NH
P460$mM NH
wild-type N. europaea variant: 4.5 ꢁ 0.1 mM DCPIP$mM cyt
2
OH with a specic activity of 2.1 ꢁ 0.1 mM DCPIP$mM cyt
ꢀ1 ꢀ1
2
OH$min . This value is ca. half of that of the
ꢀ
1
ꢀ1
ꢀ1
P460$mM
NH
2
OH$min
(Fig. 4). This observation was
corroborated by GC analysis; under turnover conditions,
Ala131Glu AL212 cyt P460 will stoichiometrically convert
NH OH to N O much like that of cyt P460 from N. europaea. WT
2 2
2
Fig. 5 Steady-state NH OH oxidation activity plot for all investigated
Fig. 4 UV-vis absorption traces showing reactions of 12 mM WT N. sp. cyt P460 variants. Assay conditions were 1 mM cyt P460, 6 mM phen-
AL212 cyt P460 (a) or AL212 Ala131Glu (b) with 10 mM NH
mM DCPIP. Reactions were carried out at 25 C in 50 mM sodium ranged from 0–20 mM. Assays were carried out anaerobically in
phosphate (pH 8.0). Reactions were initiated after collection of red 50 mM sodium phosphate, pH 8.0, at 25 C. Each data point is the
2
OH and 70 azine methosulfate (PMS), and 70 mM DCPIP. NH
2
OH concentrations
ꢂ
ꢂ
initial traces by addition of NH
collected at 15 s intervals. (c) Represents a kinetic trace of the absor- dard deviation. The data series in black represents NH
bance of DCPIP at 605 nm for both WT AL212 and Ala131Glu under the rates of DCPIP consumption under enzyme-free but otherwise iden-
2
OH, with subsequent gray scans average of at least three trials, with error bars representing one stan-
2
OH-dependent
conditions in (a) and (b), with scans every 0.5 seconds.
tical conditions.
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AL212 cyt P460 shows a basal level of N
2
O production attributed adduct, and we attributed its presence to possible O
2
contam-
to background consumption of NH OH by DCPIP (Fig. 6).
ination during sample freezing, which would react with the
2
III
7
22,26
To explore the role of the residue at position 131 in NH OH Fe –NH OH adduct to form the {FeNO} intermediate.
oxidation catalysis, we generated several other Ala131X variants Interestingly, we do not observe this {FeNO} contaminant in
of AL212 cyt P460: Ala131Gln, Ala131Asp, and Ala131Leu. None any of the other variants despite maintaining an identical
2
2
7
of these variants exhibited NH
basal levels using DCPIP as oxidant (Fig. 5). This emphasizes the catalytically active variants, intentionally exposing the Fe –
the importance of a Glu residue in the outer-sphere of the heme NH OH adducts of the catalytically-incompetent variants to O
2
OH oxidation activity above preparative method across the series. Moreover, in contrast to
III
2
2
7
P460 cofactor for catalytic competence and selectivity. We does not result in an increase of the {FeNO} signal. These
hypothesize that the inactivity of the Ala131Asp variant is due to results provide further evidence that a properly-positioned,
the inability of the shorter side chain to effectively interact with basic residue in the distal pocket of a cyt P460 is required for
bound NH OH. In further accord with our hypothesis, Glu97Ala NH OH oxidation catalysis.
2
2
2
substitution obviates NH OH oxidation in the N. europaea
variant (Fig. 5). We note that these additional variants are all
III
6
Structural studies of AL212 cyt P460 Ala131X variants
able to support Fe –NH
with either NH
additional variants show similar UV-vis absorption, NH
2
OH and {FeNO} adducts when treated
2
OH or an NO-donor, respectively. All of these That glutamine substitution fails to activate AL212 cyt P460 for
OH/NO NH OH oxidation, despite serving as an essentially iso-
reduction potentials to the two WT structural, polar surrogate for glutamate, suggests that
proteins (Fig. S4† and Table 1).
hydrogen bonding to NH OH is insufficient to promote catal-
2
2
II/III
binding affinities, and Fe
2
III
EPR spectra were obtained for the resting Fe forms of each ysis. Rather, a suitably positioned basic sidechain is required
new cyt P460 variant (Fig. 7, Table 1). All exhibit effectively axial for activity. To ensure that Gln131 can interact with Fe-bound
high-spin (S ¼ 5/2) spectra. Interestingly, the spectra of all NH OH but that this interaction is insufficient for activity, we
2
AL212 variants contain two spin systems, although the abun- undertook structural studies of the AL212 cyt P460 Ala131Glu
dances of the two components differ in each variant. We and Ala131Gln variants (Table 2). In addition, we were able to
III
2
hypothesize that these two components arise due to the pres- obtain structures of the AL212 cyt P460 Ala131Gln Fe –NH OH
6
ence of multiple conformations of a Phe side-chain in the distal and the Ala131Glu {FeNO} adducts. The global structures of all
III
NH OH/NO binding pocket (vide infra).
variants including the resting Fe WT AL212 cyt P460 are
2
The X-band EPR spectra of the NH OH-bound forms of each superimposable, with the largest pairwise RMSD values being
2
III
III
˚
variant are characteristic of low-spin Fe (S ¼ 1/2). Each Fe – 0.294 A between WT AL212 and Ala131Gln. As expected, the
NH OH variant presents a single spin system, with spin largest structural deviations are encountered in the distal
Hamiltonian parameters similar to WT N. europaea cyt P460 pocket above the heme P460 cofactor (Fig. S6†).
2
III
Fe –NH
2
OH adduct (Fig. 7). The EPR spectrum obtained for
The active site structures of the Ala131Glu and Ala131Gln
OH species for the AL212 Ala131Glu cyt P460 variants show that both residues at position 131 are positioned
variant also shows trace (ca. 5% abundance) amounts of an to participate in hydrogen bonding interactions with Fe-bound
III
the Fe –NH
2
7
{
FeNO} intermediate. We had observed this species previously substrate. This point is made clearest by the NH OH-bound
when characterizing the N. europaea cyt P460 Fe –NH OH structure of the Ala131Gln variant (Fig. 8), which demon-
2
III
2
strates that amino acids with side-chains isostructural to Glu
placed at position 131 can interact directly with bound NH OH.
2
The Fe–N bond distances for the Ala131Gln–NH OH are longer
2
˚
than anticipated (ꢃ2.5–2.7 A instead of the anticipated distance
˚
closer to 2.0 A), but we attribute this to the resolution of the
structure, at which the e.s.d. in distances is estimated to be 0.4
A˚ , or the possibility that photoreduction leading to a ferrous
center has occurred. Regardless, the composite omit map
reveals electron density consistent with a bound NH
2
OH
(Fig. S4†). Though we were unable to obtain quality data for
a structure of Ala131Glu soaked with NH OH, we anticipate
2
III
2
a high degree of similarity of the Fe –NH OH adducts based on
their effectively identical EPR spectra (Fig. 7).
Comparing the active sites of the Ala131Glu/WT AL212 and
2
Ala131Gln with NH OH bound, it is possible to see molecular
motions which may help to accommodate substrate binding
and enforce the involvement of residue 131. Specically, with
no substrate bound, Phe76 sits directly above the P460 cofactor.
Fig. 6 Plot of N
by AL212 cyt P460 variants. Assay conditions were 5 mM cyt P460,
mM DCPIP, and NH OH concentrations ranging from 0–1 mM.
Assays were carried out anaerobically in 200 mM HEPES, pH 8.0. Each
data point is the average of at least three trials, with error bars repre-
senting one standard deviation.
2 2
O production as a function of NH OH concentration
1
2
When NH OH binds, this Phe76 can reorient as is seen in the
2
Ala131Gln structures. The residue in position 131 (Gln, in this
case) can also then reposition to interact with the bound
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Fig. 7 EPR spectra of cyt P460 variants. (a) 10 K X-band (9.40 GHz) EPR spectrum (red) and simulated spectra (component 1 in black and
III
III
III
component 2 in grey) of Fe WT N. sp. AL212 cyt P460, Fe Ala131Glu, Fe Ala131Gln recorded at 633 mW microwave power. (b) EPR spectrum of
NH OH-bound Ala131Glu, Ala131Gln, and N. europaea cyt P460 under the same conditions. *Indicates the presence of contamination from the
FeNO} intermediate in both Ala131Glu and N. europaea cyt P460.
2
7
{
2
substrate (Fig. 8 and Scheme 1). This is reminiscent of In the NH OH-soaked structures of NeHAO and kustc1061, this
a conserved, carboxylate-containing Asp residue in N. europaea aspartate and a nearby histidine residue are shown to interact
HAO (NeHAO) as well as in the multi-heme, P460-containing with the bound substrate. The authors suggested, in fact, that
27
anammox enzyme kustc1061 from Kuenenia stuttgartiensis.
these residues likely participate in shuttling protons during
Table 2 X-ray data collection and crystal structure refinement statistics
III
III
Fe Ala131Glu
Ala131Glu–NO
Fe Ala131Gln
2
Ala131Gln–NH OH
Wavelength ( A˚ )
Temperature (K)
Space group
0.979
100 K
0.979
100 K
0.979
100 K
0.979
100 K
P12 1
1
48.0
80.9
120.6
90.0
1
P12 1
P12
1
1
P12
1
1
˚
a (A)
48.1
80.4
120.0
90.0
48.7
80.0
119.1
90.0
48.0
80.9
120.6
90.0
˚
b (A)
˚
c (A)
a (deg)
b (deg)
95.9
92.6
96.0
96.0
g (deg)
90.0
90.0
90.0
90.0
Reections
Number of
62 608
59 530
63 916
60 759
39 644
38 189
41 095
39 122
reections in Rwork set
Number of
3078
3157
1455
1973
reections in Rfree set
Resolution ( A˚ )
119.6–1.97
3.7 (78.8)
5.3 (111)
0.99 (0.72)
96.1 (84.9)
1.9 (1.9)
32.0 (1.1)
20.6 (43.4)
24.9 (48.4)
48.6–1.97
3.0 (84.5)
4.3 (120)
0.93 (0.68)
98.8 (96.6)
2.0 (1.9)
38.5 (0.9)
20.8 (41.6)
24.0 (45.0)
67.1–2.30
4.7 (118)
6.6 (98.8)
0.87 (0.66)
96.7 (98.3)
2.0 (2.0)
28.0 (2.1)
24.2 (33.2)
29.9 (41.5)
29.4–2.25
5.5 (115)
7.3 (165)
0.99 (0.59)
94.8 (57.8)
1.9 (1.8)
13.6 (0.5)
19.5 (34.9)
24.2 (40.0)
R
R
merge (%)
meas (%)
CC1/2
Completeness (%)
Redundancy
I/s (I)
R
R
work
free
RMSD from ideality
Bonds ( A˚ )
Angles (deg)
Average B factors (A )
0.01
1.04
53.0
0.01
1.01
57.1
0.01
1.07
50.4
0.01
1.02
52.4
2
˚
Ramachandran plot
Allowed regions (%)
Disallowed regions (%)
PDBID
100
0
6EOX
100
0
6E17
100
0
6EOZ
100
0
6EOY
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Fig. 8 Cyt P460 active site views showing orientation of residue 131. (a) Comparison of 1.97 A˚ structure of Ala131Glu (white, PBDID: 6EOX) with
2
.30 A˚ structure of Ala131Gln (green, PDBID: 6EOZ) active sites with Phe76 and residue 131 highlighted in each. (b) 2.25 A˚ structure of Ala131Gln
17
2
with NH OH bound (PDB ID: 6EOY, chain A). (c) 1.97 A˚ structure of Ala131Glu with NO bound (PDB ID: 6E17 10 , chain A).
attainable, as these residues were already in an ideal confor-
mation to accept substrate. From the EPR, it is unlikely that
O is bound in the Ala131Gln cyt P460 as it is also high spin,
H
2
though the distribution of the two components in all AL212
variants seems to be related to the mutation, and, thus, may be
related to the relative orientation preference of Phe76/
residue131. That is, where WT AL212 is almost entirely
component 1, which would correspond to Phe76 sitting directly
over the Fe, Ala131Glu is more of a mixture of the two, and is
suggestive of dynamics in which the Glu residue can occasion-
ally push the Phe76 out of the way and reorient towards the Fe
center (component 2). The EPR spectrum obtained for
Ala131Gln comprises almost entirely component 2, in accord
with the crystal structure. The dynamics of this capping Phe
residue, as well as its role in modulating substrate/product
binding affinities will be addressed in subsequent work.
Scheme 1 Proposed N. sp. AL212 Ala131Glu cyt P460 molecular
motions accompanying substrate binding.
27
catalysis. This further supports the notion that carboxylate-
containing residues are important for heme P460 NH OH
2
oxidation catalysis. Due to the chemical differences in the side-
chains of alanine and glutamine, we hypothesize that this residue
may serve as a proton relay during proton-coupled electron
transfer events in the cyt P460 catalytic cycle. This would also
explain why the corresponding Ala131Gln variant is not able to
2
Despite our inability to obtain a structure of NH OH-bound
III
2 2
oxidize NH OH, despite being able to stabilize the Fe –NH OH
Ala131Glu, we were successful in determining this variant's
structure with NO bound. Though crystals of Ala131Glu were
adduct. Though structurally very similar to glutamate, glutamine
is far less basic. Thus, despite Gln131 being able to hydrogen-
6
soaked in an NO-donor (leading to an {FeNO} ), the NO bond
bond with NH OH, it is unable to accept protons during catal-
2
distances and angles (Table 3) in some chains agree more
ysis, which is required for turnover. This implies that aer
7
26,31,32
closely with an {FeNO} conguration.
This suggests that
NH
the reaction to proceed. Based on the position of the NH
the crystal structure, the rst proton to be abstracted is likely
from the N atom of NH OH. This would lead to an NHOH radical
2
OH binding, a step involving proton transfer is necessary for
6
perhaps the {FeNO} unit was photoreduced upon data collec-
2
OH in
tion, as has been proposed for similar heme-nitrosyl crystal
33,34
structures in the NO-sensing protein nitrophorin.
Given that
2
the bond lengths and angles are also not the same in all chains,
it may suggest that more photoreduction occurred at one site
than at the other, or that the resolution is not high enough to
species analogous to that proposed during turnover of cyto-
chrome P450 nitric oxide reductase (P450nor), which reduces two
28–30
molecules of NO to N O in denitrifying bacteria and fungi.
If
6
7
2
discriminate between {FeNO} and {FeNO} intermediates.
true, this provides an interesting link between an enzyme that
Additionally, the Fe–N distances are much shorter than antici-
produces N
produces N
2
O through oxidation (cyt P460) and one that
O through reduction (P450nor). Specic details
31
pated. This is also likely a consequence of the modest reso-
2
lutions of the structures and not a meaningful reection of the
bond distances. Alternative structural techniques such as
extended X-ray absorption ne structure (EXAFS) coupled with
vibrational studies achieved using either conventional (FTIR or
resonance Raman) or nuclear resonance approaches (NRVS) will
be required to better probe the structure and electronics of
these nitrosyl species. However, the present structures suffice to
explore the outer coordination sphere. Case in point, it is clear
that Glu131 does not interact with the bound NO, however this
may not represent the conformation of the residue when the
concerning the nature and sequence of proton- and electron-
transfers involving NH OH are beyond the scope of the present
2
work, however, and will be explored in detail in a subsequent
kinetics and computational study.
III
Interestingly, the resting Fe form of Ala131Gln also has the
Phe76 residue rotated away from the heme, but in this case the
Gln131 sidechain is oriented inward towards the heme. While it
is unclear why the Gln and not the Glu variant prefers this
orientation in the crystal structure, it could explain why
a
2
NH OH-bound Ala131Gln structure was more easily
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Table 3 Bond distances and angles for substrate-bound crystal structures
Ala131Glu–NO
2
Ala131Gln–NH OH
˚
Fe–N distance in A (chain A, B, C, D)
Fe–N–O angle in deg. (chain A, B, C, D)
1.50, 1.39, 1.57, 1.78
146.36, 152.19, 134.99, 108.40
2.70, 2.75, 2.70, 2.70
121.22, 133.54, 141.47, 137.43
6
{
FeNO} is formed during NH
2
OH oxidation instead of when residue in position 131, as shown in the present work. This
theory accords with the evolutionary trajectory of HAO into
exogenous NO is added.
oxidative chemistry. HAO belongs to a larger family of multi-
heme c enzymes, including hydrazine oxidoreductase (HZO),
Functional diversity within the cyt P460 family
octaheme cytochrome c nitrite reductase (ONR), and penta-
heme cytochrome c nitrite reductase (NrfA). It is believed that
the presence of additional protein cross-links to the catalytic
heme are what distinguish enzymes with oxidative chemistries
The observation of a wild type cyt P460 that is unable to oxidize
NH OH forces us to reconsider the native function of this
2
enzyme and to consider the possibility of different classes
within the cyt P460 family that are tuned toward different
substrate specicities. Prior density functional theory analysis
showed that p-donation from the Lys N of the hallmark cyt P460
cross-link raises the energy of orbitals associated with the P460
36,37
from those with reductive chemistries.
Comparative
homology of currently annotated cyt P460 sequences would
suggest distinct classes of cyt P460 which contain different
residues (e.g. glutamate/aspartate, alanine, phenylalanine,
Fig. 9) in this crucial second sphere position. In fact, the
Nitrosomonas sp. AL212 genome contains two cyt P460
sequences (Fig. 9), one that contains Ala131 (the focus of this
study) and one that is predicted to have a glutamate in this
position (though the latter also contains a CXXCH heme-
binding motif different from the CAACH motif observed in
20
cofactor. We postulated that the ability of cyt P460 to directly
oxidize Fe-bound NH OH could be due to the higher energy
heme P460 orbitals imposed by the cross-link, which would
allow for increased NH OH character in the singly occupied
molecular orbital. This effect, however, does not have to be
specic for NH OH-oxidation. That is, the characteristic heme
2
2
2
P460 cross-link denes the oxidative capabilities of the cyt P460
enzyme, and the substrate preference/specicity could be tuned
by residues in the outer coordination sphere, such as the
38
the former AL212 cyt P460 and N. europaea cyt P460). It is likely
that this second cyt P460 behaves in a manner similar to N.
35
Fig. 9 Sequence alignment generated using MEGA X software showing homology between cyt P460 genes from N. europaea, the two cyt
P460 sequences from N. sp. AL212, and genes predicted from mammalian pathogenic bacteria Burkholderia cepacia, Pseudomonas aeruginosa,
and Vibrio metoecus. * indicates conserved Lys cross-link, ** indicates the critical second-sphere Ala/Glu/Phe residue.
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europaea cyt P460 and is responsible for some of the N
2
O
used resources of the Advanced Photon Source, a U.S. DOE
produced by the organism, which may be a means by which Office of Science User Facility operated for the DOE Office of
39–41
NH OH or NO burdens are alleviated.
The function of the Science by Argonne National Laboratory under Contract No. DE-
2
presently studied, catalytically-inactive cyt P460 in AL212 is thus AC02-06CH11357. This work is also based upon research con-
mysterious. This may allude to distinct groups of cyt P460 that ducted at the Cornell High Energy Synchrotron Source (CHESS),
perform oxidative chemistry but are tuned for different which is supported by the National Science Foundation under
substrates. Of course, we acknowledge that there are many award DMR-1332208, using the Macromolecular Diffraction at
differences among these cyt P460 genes aside from the pre- CHESS (MacCHESS) facility, which is supported by award GM-
dicted residue in this specic position, however, this study 103485 from the National Institute of General Medical
suggests that cyt P460 enzymes are capable of functions aside Sciences, National Institutes of Health. We thank Mike Fenwick
from NH OH oxidation, hence their appearance in the genomes for assistance with X-ray data collection. EPR measurements
2
18,19
of organisms that are not AOB.
The phylogenetics of the cyt were performed at the National Institutes of Health (NIH)
P460 family afford a prolic ground for subsequent studies.
ACERT with assistance from Boris Dzikhovsky. ACERT is sup-
ported by NIH/National Institute of General Medical Sciences
(NIGMS) under award number P41 GM103521.
Conclusions
Despite having heme P460 cofactors with identical spectro-
scopic, electrochemical, and substrate binding properties, N.
europaea cyt P460 is competent for NH OH oxidation and N O
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