Structural basis of biological no generation by octaheme oxidoreductases

Article abstract of DOI:10.1074/jbc.M113.525147

Nitric oxide is an important molecule in all domains of life with significant biological functions in both pro- and eukaryotes. Anaerobic ammonium-oxidizing (anammox) bacteria that contribute substantially to the release of fixed nitrogen into the atmosphere use the oxidizing power of NO to activate inert ammonium into hydrazine (N2H4). Here, we describe an enzyme from the anammox bacterium Kuenenia stuttgartiensis that uses a novel pathway to make NO from hydroxylamine. This new enzyme is related to octaheme hydroxylamine oxidoreductase, a key protein in aerobic ammonium-oxidizing bacteria. By a multiphasic approach including the determination of the crystal structure of the K. stuttgartiensis enzyme at 1.8 A resolution and refinement and reassessment of the hydroxylamine oxidoreductase structure from Nitrosomonas europaea, both in the presence and absence of their substrates, we propose a model for NO formation by the K. stuttgartiensis enzyme. Our results expand the understanding of the functions that the widespread family of octaheme proteins have.

Full text of DOI:10.1074/jbc.M113.525147

Enzymology:
Structural Basis of Biological NO
Generation by Octaheme Oxidoreductases
Wouter J. Maalcke, Andreas Dietl, Sophie J.
Marritt, Julea N. Butt, Mike S. M. Jetten, Jan
T. Keltjens, Thomas R. M. Barends and Boran
Kartal
J. Biol. Chem. 2014, 289:1228-1242.
doi: 10.1074/jbc.M113.525147 originally published online December 3, 2013
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THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 289, NO. 3, pp. 1228–1242, January 17, 2014
2014 by The American Society for Biochemistry and Molecular Biology, Inc. Published in the U.S.A.
©
Structural Basis of Biological NO Generation by Octaheme
□
S
*
Oxidoreductases
Received for publication, October 11, 2013, and in revised form, November 26, 2013 Published, JBC Papers in Press, December 3, 2013, DOI 10.1074/jbc.M113.525147
1,2 §1 ‡ʈ
‡
¶
¶
‡
Wouter J. Maalcke , Andreas Dietl , Sophie J. Marritt , Julea N. Butt , Mike S. M. Jetten , Jan T. Keltjens ,
§
3
‡4
Thomas R. M. Barends , and Boran Kartal
From the Department of Microbiology, Institute for Water and Wetland Research, Radboud University Nijmegen,
525AJ Nijmegen, The Netherlands, the Department of Biomolecular Mechanisms, Max-Planck Institute for Medical Research,
9120 Heidelberg, Germany, the Centre for Molecular and Structural Biochemistry, School of Chemistry and School of Biological
Sciences, University of East Anglia, Norwich Research Park, Norwich NR4 7TJ, United Kingdom, and Department of Biotechnology,
Delft University of Technology, 2628 BC Delft, The Netherlands
‡
§
6
6
¶
ʈ
Background: Multiheme proteins have crucial roles in diverse nitrogen cycle processes.
Results: The kustc1061 octaheme protein from anaerobic ammonium-oxidizing (anammox) bacteria specifically oxidizes
hydroxylamine to NO.
Conclusion: Enzyme specificity derives from subtle amino acid changes near the P catalytic heme.
4
60
Significance: The presence of kustc1061 homologs in anammox and other bacteria enables the detoxification of hydroxylamine,
thereby generating NO for respiratory purposes.
Nitric oxide is an important molecule in all domains of and oxidation of inorganic nitrogen compounds (1–7). Anaer-
life with significant biological functions in both pro- and obic ammonium-oxidizing (anammox) bacteria use the oxidiz-
eukaryotes. Anaerobic ammonium-oxidizing (anammox) bacte- ing power of NO to activate inert ammonium into hydrazine
ria that contribute substantially to the release of fixed nitrogen (N H ) (8). In all these processes, NO is synthesized by specific
2
4
into the atmosphere use the oxidizing power of NO to activate enzymes. Here, we describe an enzyme previously (8) identified
inert ammonium into hydrazine (N H ). Here, we describe an as kustc1061 from the anammox bacterium Kuenenia stutt-
2
4
enzyme from the anammox bacterium Kuenenia stuttgartiensis gartiensis that makes NO from hydroxylamine (NH OH)
2
that uses a novel pathway to make NO from hydroxylamine. (Equation 1). This enzyme is related to hydroxylamine oxi-
5
This new enzyme is related to octaheme hydroxylamine oxi- doreductase (HAO) of aerobic ammonium-oxidizing bacteria
doreductase, a key protein in aerobic ammonium-oxidizing bac- (9). In aerobic ammonium-oxidizing bacteria, HAO catalyzes
Ϫ
teria. By a multiphasic approach including the determination of the oxidation of hydroxylamine to nitrite (NO ), the second
2
˚
the crystal structure of the K. stuttgartiensis enzyme at 1.8 A step in the aerobic oxidation of ammonium (Equation 2). Sev-
resolution and refinement and reassessment of the hydroxyl- eral properties of this enzyme have been elucidated including
amine oxidoreductase structure from Nitrosomonas europaea, the determination of a crystal structure of the Nitrosomonas
both in the presence and absence of their substrates, we propose europaea protein (NeHAO) (9–13). Each subunit of the
a model for NO formation by the K. stuttgartiensis enzyme. Our homotrimeric NeHAO harbors eight c-type hemes for catalysis
results expand the understanding of the functions that the wide- and electron transfer, which are involved in the oxidation of its
spread family of octaheme proteins have.
substrate. A distinguishing feature of NeHAO is the covalent
attachment of the catalytic heme (fourth) of one subunit to a
4
91
tyrosine residue (Tyr ) of the adjacent subunit in the trimer
Nitric oxide has a multitude of roles in cell-cell communica- (11, 13).
tion, in microbial defense, and as a metabolite in nitrogen cycle
microorganisms, where it is an intermediate in the reduction
ϩ
Ϫ
NH OH ¡ NO ϩ 3H ϩ 3e ͑E Ј ϭ Ϫ0.030 V) (Eq. 1)
2
0
Ϫ
2
ϩ
Ϫ
NH OH ϩ H O ¡ NO ϩ 5H ϩ 4e ͑E Ј ϭ ϩ0.065 V)
2
2
0
*
This work was supported by Netherlands Organization for Scientific
Research Darwin Grants 142.16.1201 (to W. J. M.) and VENI 863.11.003 (to
B. K.), by European Research Council Advanced Grant 232937 (to M. S. M. J),
by the Max-Planck Society, and by UK Biotechnology and Biological Sci-
ences Research Council Grant BB/G009228 (to S. J. M.).
The atomic coordinates and structure factors (codes 4N4J, 4N4K, 4N4L, 4N4M,
N4N, and 4N4O) have been deposited in the Protein Data Bank
http://wwpdb.org/).
(Eq. 2)
HAO-like proteins are not unique to aerobic ammonium-
oxidizing bacteria. A BlastP search reveals hundreds of octa-
heme proteins from a wide range of microorganisms that are
homologous to NeHAO. Anammox bacteria, such as K. stutt-
gartiensis, which were shown to contribute substantially to
nitrogen release into the atmosphere (14), harbor no less than
4
(
□S
This article contains supplemental Fig. S1.
Both authors contributed equally to this work.
1
2
Present address: Institut f u¨ r Technische Biochemie, Universit a¨ t Stuttgart,
Allmandring 31, 70569 Stuttgart, Germany.
To whom correspondence may be addressed. Tel.: 496221486508; Fax:
96221486585; E-mail: Thomas.Barends@mpimf-heidelberg.mpg.de.
To whom correspondence may be addressed. Tel.: 31243652557; Fax:
1243652830; E-mail: kartal@science.ru.nl.
3
4
5
4
The abbreviations used are: HAO, hydroxylamine oxidoreductase; NeHAO,
N. europaea HAO; FL2E, 2-{4,5-bis[(6-(2-ethoxy-2-oxoethoxy)-2-methyl-
quinolin-8-ylamino)methyl]-6-hydroxy-3-oxo-3H-xanthen-9-yl}benzoic acid.
3
1
228 JOURNAL OF BIOLOGICAL CHEMISTRY
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NO Generation by Octaheme Oxidoreductases
1
0 divergent HAO paralogs. In fact, one of these (kustc0694 in tion (20 ml) was eluted by isocratic washing with 200 mM NaCl
K. stuttgartiensis) is dedicated to the oxidation of hydrazine, the in the Tris-HCl buffer. This fraction was desalted in the above
N -forming reaction in these organisms (8). Octaheme proteins potassium P buffer and concentrated to 5 ml using 100-kDa
2
i
from other organisms reduce nitrite to ammonium (15, 16). molecular mass cutoff spin filters (Vivaspin 20; Sartorius Ste-
Apparently, various types of HAO are capable of taking the dim Biotech, Aubagne, France). The concentrated fraction was
nitrogen atom through all its oxidation states between Ϫ3 loaded onto 10-ml column packed with Ceramic Hydroxyapa-
(
ammonium) and ϩ3 (nitrite), both in oxidative and reductive tite (Bio-Rad) that had been equilibrated by washing with five
directions. The presence of the tyrosine-dependent covalent column volumes of potassium P buffer. After application of the
i
linkage between different subunits seems to be a distinctive sample, the column was eluted with a 120-ml linear gradient of
feature in governing reactions into an oxidative direction (17). 20–500 mM potassium P (pH 7). Hydroxylamine oxidizing
i
These diverse catalytic potentials could very well make HAO- activity eluted as a prominent bright red symmetrical peak at 80
like proteins central but thus far overlooked catalysts in biogeo- mM potassium P . This peak (12 ml) was collected, desalted in 20
i
chemical nitrogen cycle processes. In addition to the primary mM potassium P buffer (pH 7), and concentrated as above to
i
Ϫ1
question of which reactions are performed by various HAO- ϳ2.8 mg of protein ml . Purity was checked by nondenatur-
related proteins, an even more intriguing issue is how these ating PAGE and SDS-PAGE (19). The identity of kustc1061 was
variants are tuned to their specific functions.
established by MALDI-TOF analysis (see below). By the proce-
Presently, it is unknown which structural features deter- dure outlined, kustc1061 was purified 5.3-fold in 31% yield.
mine the differences in reaction specificities of HAO-like Both the yield and purification factor will likely be underestimates
proteins. Furthermore, these differences cannot be explained because crude extract contained substantial hydroxylamine oxi-
by sequence analyses and structural modeling, which are ham- dizing activity derived from hydrazine synthase (8).
pered by the lack of structural and biochemical information. To
N. europaea ATCC 19718 was cultured, and NeHAO was
address these questions, we studied the catalytic and redox purified essentially as described by Hooper and Nason (20). 110
properties of the HAO-like protein kustc1061 of K. stutt- liters of a N. europaea culture were grown at room temperature
gartiensis. Further, we determined the high resolution structure in mineral medium (ATCC medium 2265) in 20-liter carboys,
of both the as-isolated kustc1061 and crystals soaked with sub- which were vigorously sparged with air. The pH of the medium
strates. In parallel and independent from a recent refinement was regularly adjusted to 8 by adding 20% (w/v) Na CO . The
2
3
(
12, 13), we reassessed the structure of NeHAO not only in the cells were harvested after 6 days (12 g of wet weight), frozen in
absence but also in presence of substrates. The results of this liquid nitrogen, and stored at Ϫ80 °C. The cells were resus-
study shed light on determinants in a widespread group of pended in 50 mM Tris-HCl (pH 7.5; buffer A) and disrupted by
homologs about which very little is known at the biochemical sonication, and the cell-free extract was fractionated by ammo-
level.
nium sulfate precipitation followed by subsequent DEAE-Sep-
harose (Merck) and ceramic hydroxyapatite (Bio-Rad) liquid
chromatography. After exchange to buffer A using a PD-10
EXPERIMENTAL PROCEDURES
Enzyme Purification and Identification—Kustc1061 was desalting column (GE Healthcare), the 35–70% ammonium sul-
purified from a K. stuttgartiensis enrichment culture (ϳ95% fate fraction was loaded onto a 30-ml DEAE-Sepharose column,
pure) that was grown continuously as planktonic cells in a 10-li- which was eluted with a linear gradient of 0–400 mM NaCl in
ter membrane bioreactor using methods described by Kartal et buffer A. The red fractions eluting at 200 mM NaCl were com-
al. (18). Purification typically used 4 liters of cells (A ϭ ϳ1) bined, buffer-exchanged to 20 mM potassium P buffer (pH 7.5),
600
i
that were collected by centrifugation (8,000 ϫ g, 15 min). This and loaded onto a 5-ml hydroxyapatite column, which was
and all following steps, except for FPLC, were done at 4 °C. eluted with a gradient of 20–500 mM potassium phosphate (pH
Pelleted cells were resuspended in 5 ml of 20 mM potassium P 7.5). NeHAO and Ne1300 co-eluted at ϳ250 mM potassium
i
buffer (pH 7) and broken by three subsequent passages through phosphate. These fractions were concentrated and buffer-ex-
a French pressure cell operating at 138 MPa. The lysate was changed to 25 mM HEPES-KOH (pH 7.5) containing 25 mM
incubated with 1% deoxycholate (w/v) on a rotating incubator KCl. The identity of the proteins was confirmed by MALDI-
(
20 rpm) for 1 h. After incubation, cell debris was removed by TOF mass spectrometry after tryptic digest from SDS-PAGE
centrifugation (3,000 ϫ g, 15 min), and the supernatant was gel slices.
subjected to ultracentrifugation (150,000 ϫ g, 1 h) in a Discov-
Analytical Ultracentrifugation—Sedimentation velocity and
ery 100 ultracentrifuge equipped with a T-1270 rotor (Sorvall, equilibrium ultracentrifugation was performed in a Beckman
Newton, CT) to pellet cell membranes. The deep dark red XL-I Proteomelab ultracentrifuge, using 1.2-cm path length
supernatant represented the crude extract. Kustc1061 was cells at 20 °C. The protein was dissolved in 25 mM HEPES-KOH
brought to homogeneity in a two-step FPLC procedure using an (pH 7.5), 25 mM KCl to an absorbance at 280 nm of less than 0.5
¨
Akta purifier (GE Healthcare). Columns were eluted at 2 ml (for 1.2 cm path length). The speeds used were 9,000 and 12,000
Ϫ1
min ; the eluate was monitored at 280 nm and collected in rpm in an An60Ti rotor. Equilibrium data were analyzed using
2
-ml fractions. In the first step, crude extract was applied to a SEDFIT (21).
column packed with 30 ml of Q Sepharose XL (GE Healthcare)
Spectrophotometric Enzyme Assays—By routine, reactions
and equilibrated with two column volumes of 20 mM Tris-HCl were followed at 37 °C by measuring the reduction of bovine
Ϫ1
Ϫ1
buffer (pH 8). After washing the column with two column vol- cytochrome c at 550 nm (⌬⑀ ϭ 19,600 M cm ) (22) in a
5
50
umes of this Tris-HCl buffer, a hydroxylamine-oxidizing frac- Cary 50 spectrophotometer (Agilent, Santa Clara, CA). Reac-
JANUARY 17, 2014•VOLUME 289•NUMBER 3 JOURNAL OF BIOLOGICAL CHEMISTRY 1229

NO Generation by Octaheme Oxidoreductases
␥
␤
␣
␴
1
230 JOURNAL OF BIOLOGICAL CHEMISTRY
VOLUME 289•NUMBER 3•JANUARY 17, 2014

NO Generation by Octaheme Oxidoreductases
FIGURE 1. Hydroxylamine and hydrazine oxidation by kustc1061 and NeHAO. A, cytochrome c reduction (50 ␮M) catalyzed by kustc1061 (200 ng) in the
presence of 1.5 ␮M hydroxylamine (blue) or 1.5 ␮M hydrazine (red). The oxidation of hydroxylamine (1.5 ␮M) coupled to the reduction of cytochrome c (50 ␮M)
by NeHAO (30 ng) is represented by the green curve. Reactions were followed and quantified by measuring the increase in the absorption at 550 nm (⌬⑀
ϭ
5
50
Ϫ1
Ϫ1
1
2
9,600 M cm ) as the result of cytochrome c reduction. B, hydroxylamine oxidation (9 ␮M) by 1.4 ␮g of kustc1061 (blue) and hydrazine oxidation (9 ␮M) by
0 ␮g of kustc1061 (red) recorded by measuring the reduction of cytochrome c at 550 nm (⌬⑀550 ϭ 19,600 M cm , right axis). C, NO production from
Ϫ1 Ϫ1
hydroxylamine (8 ␮M) by kustc1061 (150 ng) in the presence of 50 ␮M cytochrome c. NO formation was followed by recording the increase in fluorescence as
the result of the formation of the nitrosylated derivative of Cu(II)FL2E and was quantified from a calibration curve prepared from known NO stock solutions. D,
hydroxylamine oxidation (2 ␮M) recorded by the increase in fluorescence resulting from the formation of a nitrosylated derivative of the NO-probe 4-amino-
5
30 15 15 15
versus cytochrome c reduction. E, accumulation of N during [ N]hydrazine oxidation.
H
c was determined as in A. NO in C and D was quantified from calibration curves prepared from known NO stock solutions.
tion rates were determined from the initial linear portion of the ing 50 ␮M cytochrome c with 20 ␮M ascorbic acid, giving 40 ␮M
progress curves employing the Cary 50 software package. For reduced cytochrome c.
enzyme kinetics, initial reaction rates were fitted by nonlinear
Nitrite reduction with reduced methyl viologen as electron
regression analysis (Origin 8.5.1; OriginLab Corporation, donor was followed by recording methyl viologen oxidation at
Ϫ1
Ϫ1
Northampton, MA) applying Michaelis-Menten equations.
600 nm (⑀ ϭ 13,700 M cm ) (23). Reduced methyl violo-
6
00
Reaction mixtures (0.4 ml) in potassium P buffer contained gen monocation radical was made by zinc reduction of a 1 mM
i
5
0 ␮M cytochrome c and an appropriate amount of enzyme. stock solution of the oxidized compound (24). The preparation
After following the absorption at 550 nm for 1 min, reactions of reduced methyl viologen and of the assay mixtures and sub-
were started by the addition of substrate (hydroxylamine or sequent enzymatic reactions were carried out in an anaerobic
hydrazine) in the requested concentration from 100 ␮M anoxic glove box (97% cytochrome, 3% H ; O Ͻ 0.2 ppm). Reduced
2
2
stock solutions. Potential inhibitors (90 ␮M NO, 5 mM phenyl methyl viologen was added to an A ϭ 1 (73 ␮M), and reac-
6
00
hydrazine) were added prior to the enzyme. An NO stock solu- tions were started by the addition of enzyme.
tion (0.9 mM) was prepared by sparging anoxic potassium P
Spectrofluorometric NO Measurements—Nitric oxide produc-
i
buffer with He/NO (50%:50%, v/v) in a butyl-rubber capped tion was measured by two different fluorescence-based methods.
serum vial for 10 min. For assays in the direction of reduced Fluorescencewasrecordedin4-mlrubber-stopperedfluorescence
cytochrome c oxidation, a stock solution was prepared by mix- cuvettes (Hellma, M u¨ llheim, Germany) in a Cary Eclipse fluorom-
JANUARY 17, 2014•VOLUME 289•NUMBER 3
JOURNAL OF BIOLOGICAL CHEMISTRY 1231

NO Generation by Octaheme Oxidoreductases
TABLE 2
Catalytic and structural properties of kustc1061 from K. stuttgartiensis, characterized orthologues from B. anammoxidans, and anammox strain
KSU1 and hydroxylamine oxidoreductase from N. europaea
Ϫ
Kinetic values are given for mammalian cytochrome c as electron acceptor. Reduced methyl viologen was used as electron donor for NO2 reduction. NR, not reported.
Property
K. stuttgartiensis
B. anammoxidans
KSU-1
N. europaea
Ϫ1
V
k
NH OH (␮mol min mgϪ1
)
4.8
21
9.6
28.5
95
3.6
26
14
47
4
max
2
Ϫ1
NH OH (s
)
15
64
19
cat
m
2
K
NH OH (␮M)
4.4
3.4
1.6
4.9
54
26
33
2
Ϫ1
Ϫ1
k
/K NH OH (s ␮M
)
2.5
1.1
3.4
18
0.57
0.54
1.1
25
cat
V
m
2
N H (␮mol min mgϪ1)
2 4
Ϫ1
Ϫ1
max
k
N H (s
)
cat
2
4
K
N H (␮M)
2 4
Ϫ1
m
k
/K N H (s ␮MϪ1
)
0.1
0.19
0.04
NR
118
53
12
cat
m
2
4
NO reduction (␮mol min mgϪ1
Ϫ
Ϫ1
)
0.18
a
0.87
b
6.18
c
2
Total size (kDa)
184
183
58
200
67
Subunit size (kDa)
Subunit composition
61.5
␣
␣
␣
␣
3
3
2
3
Catalytic heme optical maximum (nm)
References
468
468
468
463
This paper
49
50
20, 51, 52
a
b
c
Specific activity with 0.1 mM nitrite.
Specific activity with 2.5 mM nitrite.
V
max
.
eter (Agilent) set at a slit width of 5 nm and high photomultiplier
voltage. Calibration was performed in the under-mentioned reac-
tion mixtures without enzyme containing 0–13.5 ␮M of NO.
The first method used the Cu(II) complex of FL2E (2-{4,5-
bis[(6-(2-ethoxy-2-oxoethoxy)-2-methylquinolin-8-ylamino)
methyl]-6-hydroxy-3-oxo-3H-xanthen-9-yl}benzoic acid) (25)
supplied in the NO-ON nitric oxide sensing kit (Strem Chem-
icals, Newburyport, MA) as the trapping agent. Cu(II)FL2E re-
acts specifically with NO in a 1:2 stoichiometry to form a fluo-
rescent nitrosylated derivative. Assays were performed
anaerobically at room temperature. Reaction mixtures (4 ml)
were prepared in the anaerobic glove box and contained 8 ␮M
hydroxylamine, 50 ␮M cytochrome c, 0.5% (v/v) Me SO, and 10
2
FIGURE 2. Electronic absorbance spectra of kustc1061. Spectra are the fol-
lowing: as-isolated (fully oxidized) kustc1061 (80 ␮g ml ) (gray), dithionite-
reduced kustc1061 (red), and the as-isolated enzyme incubated either with
␮
M Cu(II)FL2E in 20 mM potassium P buffer (pH 7). After re-
i
Ϫ1
cording fluorescence (excitation wavelength, 480 nm; emission
wavelength, 519 nm) for 2 min, the reaction was started by 100 ␮M hydroxylamine (dark blue) or with 100 ␮M hydrazine (light blue).
adding enzyme.
The second method took advantage of the NO- and oxygen-
The identity of proteins was established by MALDI-TOF
dependent formation of nitrosylated 4-amino-5-methylamino- analysis of the trypsin-cleaved protein prepared from the gels as
2
Ј,7Ј-difluorofluorescein (Calbiochem) (26, 27). In this case, detailed elsewhere (8, 30). Spectra were evaluated by the Mas-
assays were performed aerobically. Reaction mixtures (4 ml) cot Peptide Mass Fingerprint search program (Matrix Science,
contained 2 ␮M hydroxylamine, 50 ␮M cytochrome c, 0.1% (v/v) London, UK) against the K. stuttgartiensis and the N. europaea
Me SO, and 8.25 ␮M 4-amino-5-methylamino-2Ј,7Ј-difluoro- protein databases. In case of kustc1061, 12 of 43 predicted pep-
2
fluorescein in 20 mM potassium P buffer (pH 7). Reactions were tides were retrieved (Mowse identification score, Ͼ50) (supple-
i
recorded at an excitation wavelength of 495 nm and an emis- mental Fig. S1).
sion wavelength of 518 nm and were started by the addition of
Electronic Absorbance Spectra—UV-visible spectra were
0
.6 ␮g of enzyme.
measured in 1-ml quartz cuvettes (path length, 1 cm). Samples
15
Ϫ1
Other Analytical Methods—( N-Labeled) gaseous nitrogen contained 80 ␮g ml of as-isolated fully oxidized kustc1061 in
compounds (NO, NO , N O, and N ) were determined by potassium P buffer. Spectra of the reduced kustc1061 were
2
2
2
i
GC-MS analysis of head space samples as described previously recorded immediately after adding few crystals of sodium
8). Here, reactions were performed in 3-ml Exetainers (Labco, dithionite to the solution containing oxidized enzyme. Spectra
High Wycombe, UK) closed with rubber stoppers. Reaction in the presence of substrates were made after preincubating
(
15
mixtures (2 ml) containing either 10 ␮M [ N]hydroxylamine oxidized or reduced kustc1061 solution with 100 ␮M of the
1
5
(
99% pure; Isotec, Miamisburg, OH) or 7.5 ␮M double-( N) compound to be tested (5 mM for phenyl hydrazine). NO (final
labeled hydrazine (98% pure; Cambridge Isotope Laboratories, concentration, 90 ␮M) was added from a stock solution pre-
Cambridge, UK) were prepared in the anaerobic glove box. pared as described above.
Ammonium and nitrite were measured colorimetrically
Spectroelectrochemistry—Heme reduction potentials were
according to established methods (28). Protein concentrations measured through changes in the electronic absorbance of
were measured with the Bio-Rad protein assay, based on the kustc1061 that had been adsorbed on an optically transparent
method of Bradford (29), using bovine serum albumin as a SnO electrode and then equilibrated at 4 °C at a series of
2
standard.
defined potentials following the protocol previously published
1
232 JOURNAL OF BIOLOGICAL CHEMISTRY
VOLUME 289•NUMBER 3•JANUARY 17, 2014

NO Generation by Octaheme Oxidoreductases
FIGURE 3. Protein film electrochemistry of kustc1061. A, full spectra of the oxidative titration from Ϫ490 to ϩ230 mV versus standard hydrogen electrode
(
SHE) in 20-mV steps. B, table showing the E Ј values (Ϯ 10 mV) calculated for the successive oxidations and reductions of the c-type hemes in kustc1061 and
m
the comparison to those reported for NeHAO (53, 54). Numbers in parentheses refer to the corresponding hemes in NeHAO. Note that the heme c midpoint
redox potentials in kustc1061 are generally lower, but the method applied does not allow the assignment to specific hemes, except for the catalytic heme 4.
C, spectral details of the Soret region. D, signals at 418 nm normalized to 1 and plotted against the applied potentials. E, spectral details of the heme c-␣ and -␤
bands. F, signals at 551 nm normalized to 1 and plotted against the applied potentials. G, spectral details of the P460 region. H, signals at 468 nm normalized to
1
and plotted against the applied potentials. Squares in D, F, and H represent experimental values. Solid lines in these graphs are Nernstian fits. The data in D and
F were well described by the sum of seven Nernstian contributions from isolated, single-electron (n ϭ 1) redox centers having the E Ј values listed in B (all Ϯ
m
1
0 mV). It was assumed that P460 spectral properties did not interfere significantly with the spectral changes of the Soret (C) and ␣ and ␤ (E) bands of the His/His
ligated hemes; clear isosbestic points at 435 nm and at 505 and 561 nm, respectively, supported this assumption. The variation in absorbance at 468 nm had
contributions from redox transformation of the P460 co-factor in addition to those of low spin His/His ligated hemes. The changes in absorbance caused by the
P460 co-factor were readily identified through their larger magnitude and the appearance of a peak at low potential with an isosbestic point at 455 nm. The plot
of normalized A468 nm Ϫ A455 nm was well described by a fit to the Nernst equation for an n ϭ 1 center with E Ј ϭ Ϫ300 mV.
m
JANUARY 17, 2014•VOLUME 289•NUMBER 3
JOURNAL OF BIOLOGICAL CHEMISTRY 1233

NO Generation by Octaheme Oxidoreductases
(
31). The electrode was composed of a mesoporous, nanocrys-
talline layer of SnO coated onto a glass slide (32). The electrode
2
2
(
ϳ0.7-cm geometric area) was covered with 5 ␮l of 3.5 mg
Ϫ1
ml kustc1061 in potassium P buffer and incubated on ice for
i
1
0 min. After removal of excess protein solution, the electrode
was rinsed with 50 mM MOPS buffer (pH 7) and placed in an
anaerobic 3-ml quartz cuvette (path length, 1 cm) filled with 50
mM MOPS buffer (pH 7). A platinum wire and an AgCl-coated
silver wire served as counter and reference (E Ј ϭ ϩ400 mV
0
versus standard hydrogen electrode) electrodes, respectively.
The cuvette containing the kustc1061-coated SnO electrode
2
was placed in a spectrophotometer (V650; Jasco Analytical
Instruments, Easton, MD) and connected to a potentiostat
(
PGSTAT20; Autolab, Utrecht, The Netherlands). To mini-
mize the spectral contribution from scattering by the SnO par-
2
ticles, a cuvette containing a SnO electrode that had not been
2
exposed to kustc1061 was placed in the reference beam of the
spectrometer during measurements. UV-visible spectra were
recorded after setting the potential applied to the kustc1061-
coated electrode to the desired value and allowing the protein
to equilibrate with that potential. Voltage-dependent spectral
changes were fully reproducible for at least five cycles between
Ϫ600 and ϩ400 mV (20 mV steps) in reductive and oxidative
directions. Changes in absorbance at 551 and 418 nm derived
from the heme c ␣ and Soret bands, respectively, and at 468 nm
of the P
chromophore were normalized and evaluated by
4
60
Nernstian curve fitting using Microsoft Excel and applying least
square minimizing.
Protein Crystallization and Crystal Handling—Kustc1061
was concentrated by ultrafiltration to A
Ϸ 20 (A
280,1 cm
409,1 cm
Ϸ 95) in 25 mM HEPES-KOH buffer (pH 7.5), containing 25 mM
KCl. Needle-shaped, hexagonal crystals of kustc1061 grew from FIGURE 4. X-ray crystal structure of kustc1061 from K. stuttgartiensis. A,
views of the kustc1061 trimer perpendicular to (left panel) and along the
hanging drops equilibrated against 1.2 M ammonium sulfate,
3
-foldsymmetryaxis(rightpanel). Thethreemonomersareshownindifferent
0
.1 M sodium phosphate buffer (pH 7.4). These crystals were
colors. The 24 heme groups are shown in red. B, the same views of the
soaked in reservoir solution containing 25% (v/v) ethylene gly- kustc1061 trimers, showing the outline of the protein and the ring-like
arrangement of heme groups (red). C, stereo figure, showing a close-up of
col and 10 mM HgCl before flash-cooling in liquid nitrogen.
2
the active site of kustc1061. The P460 co-factor consists of a heme c moiety
Another, cubic crystal form diffracting to much higher resolu-
451
(
white sticks) covalently bound in two places to the Tyr side chain of an
262 263
tion grew from 1.3 M ammonium sulfate, 0.05–0.1 M sodium adjacent protein monomer (blue). The conserved Asp /His is shown, as
2
27
well as His , which coordinates the heme iron as the proximal ligand. A
phosphate buffer (pH 7.4), and the detergent additive cyclo-
water molecule (red sphere) forms the distal ligand. The final, refined 2mF Ϫ
o
hexylbutanoyl-N-hydroxyethylglucamide. These crystals were DF electron density map calculated from 1.8 Å resolution data is overlaid at a
c
2
␴ contour level (blue mesh). The figures were prepared in PyMOL (47).
cryoprotected in reservoir solution with 25% (v/v) ethylene gly-
col or 30% (w/v) sucrose and then flash-cooled in liquid
nitrogen.
Swiss Light Source (Villigen, CH) at 100 K and processed with
NeHAO (A2
Ϸ 37; A
Ϸ 133) was crystallized XDS (35, 36). Statistics are reported in Table 1. A highly redun-
80,1 cm
409,1 cm
essentially as described by Cedervall et al. (12, 13). Thin oval dant, 3.5 Å resolution SAD data set was collected from a hex-
plates of up to 200-␮m longest dimension grew at 20 °C under agonal crystal at a wavelength of 1.7384 Å, in which
Al’s oil (a 1:1 mixture of paraffin oil and silicon oil) in batch AutoSHARP (37) detected seven heavy atom sites per asym-
crystallization using 42% (v/v) PEG400 and 50 mM KNO in 100 metric unit using SHELXD (38). Phasing and density modifica-
3
mM MES/NaOH buffer (pH 7.5), after 2 weeks. When flashed- tion using SOLOMON (39) resulted in an excellent electron
cooled in their mother liquor, the space group was P2 2 2 with density map into which an initial model was built, which was
1
1 1
a ϭ 142 Å, b ϭ 143 Å, c ϭ 214 Å. These crystals could also be then refined against a 2.6 Å resolution data set from a hexagonal
dehydrated by increasing the PEG400 concentration to 65% crystal collected at a wavelength of 1.000 Å. After several
(
v/v) in 5% steps, resulting in space group P2 2 2 with a ϭ 140 rounds of rebuilding in COOT (40) and refinement with
1
1
Å, b ϭ 141 Å, c ϭ 106 Å. A large improvement in diffraction REFMAC (41) and PHENIX (42), a model with excellent geometry
quality was observed upon annealing (33, 34) the flash-cooled andRfactorswasobtained(Table1). Thismodelwasusedtophase
crystals by interrupting the cryostream once for 15 s.
the high resolution data obtained from the cubic crystal form by
Data Collection and Structure Solution—Diffraction data molecular replacement with PHASER (43). Here, too, repeated
from kustc1061 were collected at the X10SA Beamline of the rounds of rebuilding in COOT (40) and refinement with REF-
1
234 JOURNAL OF BIOLOGICAL CHEMISTRY
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NO Generation by Octaheme Oxidoreductases
FIGURE 5. Views of kustc1061 and NeHAO along the 3-fold axis of the trimers. A, kustc1061, with the N-terminal domain shown in red. B, NeHAO, with the
Ne1300 polypeptide shown in orange. In kustc1061, the N-terminal domain adopts the position of Ne1300 in its complex with NeHAO.
MAC (41) resulted in an excellent model with good R factors at the X10SA Beamline of the Swiss Light Source (Villigen, CH)
(
Table 1). Geometric parameters for the modified heme co-fac- at 100 K. Difference electron density maps clearly displayed
tor were obtained from semiempirical calculations at the PM-3 extra density connected to the position where a water molecule
level carried out using HyperChem 8.0 (Hypercube Inc., is bound in the native structure, clearly showing that a larger
Gainesville, FL). Crystals of protein-ligand complexes were molecule had bound to the P co-factor.
4
60
obtained by soaking cubic crystals in artificial mother liquor
Bioinformatics—Protein sequence homology searches were
containing the respective compounds (100 mM hydroxylammo- performed against nonredundant protein databases using the
nium chloride, 10 min; 5 mM hydrazinium sulfate, 10 min; and BlastP program (44) at the NCBI website. N-terminal signal
5
0 mM phenyl hydrazine, 30 min) followed by flash-cooling in cleavage sites were predicted with SignalP 4.1 (45). Multiple
liquid nitrogen. Soaking with NO was done by incubating crys- amino acid sequence alignments were made with the ClustalW
tals in thoroughly degassed and cytochrome-saturated buffers, program (46) at the EMBL-EBI website. All structural figures
and cryoprotectants were placed inside an anaerobic glove box were prepared in PyMOL (47).
for 15 min at room temperature with ϳ4 mM of the NO donor
RESULTS
1
-(N,N-diethylamine)diazen-1-ium-1,2-diolate.
NeHAO crystals of either crystal form were annealed as
Kustc1061 Is a Specific NO-forming Hydroxylamine Oxidase—
described above, and native data were collected at PETRA-III Previously, we purified an abundant HAO-like protein from
using a Pilatus 6 M detector at 100 K and processed with XDS K. stuttgartiensis and identified this as kustc1061 (8). This pro-
(
35, 36). Molecular replacement with PHASER (43) using the tein performed the three-electron oxidation of hydroxylamine,
structure from Igarashi et al. (11) (Protein Data Bank 1FGJ) as resulting in the stoichiometric formation of NO and three mol-
the search model. During iterative rebuilding in COOT (40) ecules of reduced cytochrome c (Fig. 1, A–D, and Equation 1).
and refinement with REFMAC (41), an extra polypeptide chain In contrast and as expected, the purified NeHAO catalyzed a
became visible in the electron density maps. This protein was four-electron oxidation of hydroxylamine in accordance with
confirmed to be Ne1300 by peptide mass fingerprinting and Equation 2 (Fig. 1A). Hydroxylamine oxidation by kustc1061
was built accordingly. A ligand-coordinated structure of followed Michaelis-Menten kinetics (apparent K ϭ 4.4 Ϯ 0.9
m
Ϫ1
Ϫ1
NeHAO was obtained by performing the dehydration protocol ␮M, V
ϭ 4.8 Ϯ 0.2 ␮mol min mg protein ). NO in con-
max
in the presence of 100 mM hydroxylamine. Data were collected centrations up to 80 ␮M did not have any effect on this reaction.
JANUARY 17, 2014•VOLUME 289•NUMBER 3 JOURNAL OF BIOLOGICAL CHEMISTRY 1235

NO Generation by Octaheme Oxidoreductases
FIGURE6. Structuralsimilarityofthecatalyticfeaturesofkust1061andNeHAO. A, stereoviewofasuperpositionoftheactivesitesofkustc1061(lightgray)
and NeHAO (dark gray) soaked in hydroxylamine. B, stereo view of a superposition of kustc1061 and NeHAO based on C␣-carbons, showing only the heme
groups. The two proteins show a virtually identical arrangement of hemes.
Further, kustc1061 was also able to catalyze the four-electron ment with the theoretical mass of 184.16 kDa calculated for a
oxidation of hydrazine to N (Fig. 1, A and E), albeit with lower homotrimeric gene product, after cleavage of a predicted signal
2
affinity and maximum rate (apparent K ϭ 54 Ϯ 3.3 ␮M, peptide, harboring 24 heme c molecules in total. The resolution of
m
Ϫ1
Ϫ1
V
ϭ 1.60 Ϯ 0.05 ␮mol min mg protein ), i.e., with a the crystal structure described below confirmed the presence of
max
3
4-fold lower catalytic efficiency (k /K ) (Table 2). Kustc1061 three identical subunits, each binding eight c-type hemes.
cat
m
was capable of nitrite reduction with an artificial reductant,
The UV-visible spectrum of the as-isolated kustc1061 exhib-
reduced methyl viologen, but only with a very low rate (0.18 ited the characteristic features of low spin ferric c-heme,
Ϫ1
Ϫ1
␮
mol min mg protein ), which is also comparable to nitrite namely, a pronounced maximum at 408 nm in the Soret region
reduction by NeHAO (Table 2). In contrast to what was and a broad feature of lower intensity between 500 and 600 nm
reported for NeHAO (48), we could not measure any hydrox- (Fig. 2). Upon addition of dithionite, these features of the fully
ylamine disproportionation to ammonium and NO, nitrite, oxidized enzyme changed, giving rise to maxima at 418, 522,
N O, or N . These findings strongly suggested that the oxidation and 551 nm and at 468 nm. The maxima at 418, 522, and 551 nm
2
2
of hydroxylamine to NO was the distinctive physiological reaction were indicative of (His/His ligated) low spin ferrous c-hemes,
of kustc1061. The kustc1061 catalytic constants were in the same whereas that at 468 nm was reminiscent of a ferrous heme with
range as those reported for not well defined, homologous enzymes covalent modification by tyrosine as found for the P catalytic
4
60
of the anammox bacteria Brocadia anammoxidans and strain heme in NeHAO (11, 13, 53). Cross-linking of kustc1061 sub-
KSU-1 (Table 2) (49, 50). NeHAO oxidized hydroxylamine and units by covalent modification of the heme was supported by
hydrazine at 6- and 9-fold higher maximal rates, respectively.
the failure of the three subunits to dissociate upon denaturing
Kustc1061 Is a Multiheme Protein with a Low Redox Potential SDS-PAGE (data not shown) and confirmed by x-ray crystal-
Chromophore—Kustc1061 was purified as a protein with a lography (see below). Using protein film spectroelectrochemis-
P
4
60
molecular mass of 184.20 kDa as determined by sedimentation try, the low spin His/His-ligated hemes could be reversibly
equilibrium ultracentrifugation. This value was in excellent agree- reduced in seven consecutive one-electron reduction steps
1
236 JOURNAL OF BIOLOGICAL CHEMISTRY
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NO Generation by Octaheme Oxidoreductases
showing midpoint redox potentials at pH 7 (E Ј) ranging from
m
Ϫ10 to Ϫ410 mV versus the standard hydrogen electrode (Fig.
3
). This potential window encompassed six E Ј values for the
m
His/His-ligated hemes in NeHAO (53, 54). The P
chro-
4
60
mophore in kustc1061 displayed an E Ј of Ϫ300 Ϯ 10 mV
m
compared with E Ј ϭ Ϫ260 mV for NeHAO.
m
When as-isolated kustc1061 was incubated with its substrate
hydroxylamine, the protein became partly reduced (Fig. 2).
Comparison with protein film spectroelectrochemistry results
suggested the reduction of two hemes such that hydroxylamine
would undergo a two-electron oxidation upon binding to the
enzyme. Incubation with hydrazine also resulted in a spectrum
indicative of two-electron reduction. In contrast, NO had no
effect on the absorption spectrum of oxidized kustc1061, but it
reoxidized the reduced enzyme completely.
Kustc1061 and NeHAO Share a Common Architecture—The
high resolution x-ray crystal structure of kustc1061 was pre-
dominantly ␣-helical, with long helices running approximately
parallel to the 3-fold symmetry axis of the trimer and surround-
ing a central, water-filled cavity (Figs. 4 and 5A). The subunits
451
were linked by two covalent bonds between Tyr of one sub-
unit and heme 4 of an adjacent subunit. Heme 4 was ligated by
227
a single amino acid (His ) and could be assigned as the P
4
60
co-factor identified by UV-visible spectroscopy. The additional
seven c-type hemes had His/His ligation, so that together they
readily accounted for the additional ferric and ferrous heme
features observed in the UV-visible spectra.
Despite a sequence identity of only 26% to NeHAO (supple-
mental Fig. S1), the structures of kustc1061 and NeHAO were
similar and could be superimposed to a root mean square posi-
tional difference of 1.7 Å for 377 C␣ atoms. This conservation
also applied to the position of the heme moieties (Figs. 4–6).
One may note that the same heme configuration was observed in
other octaheme proteins and in pentaheme nitrite reductase
(
NrfA) in which the five hemes (1–5) superimpose with hemes
4
–8 of the octaheme proteins (14, 15, 55, 56). However, in the
trimeric kustc1061 and NeHAO, hemes were arranged in a ring. It
was proposed earlier that such a ring-like arrangement of single
electron carriers would enable electron transfer between the sub-
units, thus facilitating the extraction of two electrons from the
substrate in rapid succession (54), which could also be essential for
the oxidation of NH OH to NO catalyzed by kustc1061.
2
Apart from this structural similarity between kustc1061 and
NeHAO, we observed distinguishing differences between these
two proteins, notably in their N- and C-terminal parts.
Kustc1061 had a negatively charged N-terminal domain (N1;
amino acids 36–80 of the translated protein) that clung to a
positively charged amino acid stretch of an adjacent subunit
(
Fig. 5). This domain was absent in NeHAO. Instead, and in full
FIGURE 7. Stereo views of F ؊ DFc electron density maps for the sub-
stratesoaks, calculatedpriortotheinclusionoftheligandsinthemodel.
o
agreement with the structure recently published by Cedervall et
al. (13), the subunit interface in our native and complexed Positivedifferencedensity(green)iscontouredat4␴, andnegativedifference
density (red) is contoured at Ϫ4 ␴. A, kustc1061 soaked in NH OH. B,
2
NeHAO structures was occupied by a polypeptide of 69 amino
kustc1061 soaked in hydrazine. C, kustc1061 soaked in phenyl hydrazine. D,
acids, which was identified as a fragment of the hypothetical Nitrosomonas HAO soaked in NH OH.
2
protein Ne1300 (101 amino acids). However, these overall
structural differences between the two proteins did not imme-
Kustc1061 and NeHAO Have Structurally Similar P4
60
diately explain why in kustc1061 the oxidation of NH OH stops Co-factors—We determined kustc1061 crystal structures
2
Ϫ
at NO, whereas in NeHAO the reaction continues to NO .
soaked with substrates, observing binding to heme 4 (the P
2
460
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NO Generation by Octaheme Oxidoreductases
FIGURE 8. X-ray structures of the kustc1061 and NeHAO catalytic sites in crystals soaked with substrates. Close-ups of the active sites of kustc1061
soaked with hydroxylamine (A), hydrazine (B), and phenyl hydrazine (C) and of NeHAO soaked with hydroxylamine (D) are shown.
co-factor), which was apparently the active site, as in NeHAO structure of kustc1061, we observed two covalent bonds
4
51
451
(
11, 13). Considering the difference in reaction specificity, it between Tyr and the P co-factor, between a Tyr C⑀ and
4
60
451
was surprising that at first glance the active sites of kustc1061 the heme meso-carbon C5, as well as between the Tyr O␩
and NeHAO appeared highly similar as well (Figs. 6 and 7A). In and the heme carbon atom C4 (Figs. 4C, 6A, 7, A–C, and 8,
both proteins, heme 4 was covalently linked to a tyrosine from A–C). Although only the latter of these two was built in the
another subunit, causing the co-factor to be substantially ruf- previous, 2.8 Å resolution structural model of NeHAO (11),
fled. This ruffling was proposed to support electron abstraction both in our higher resolution structures and in the recent
from the substrate by the heme (11, 54). In the 1.8 Å resolution refinement (2.1 Å) (13), two bonds were clearly present (Figs.
1
238 JOURNAL OF BIOLOGICAL CHEMISTRY
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NO Generation by Octaheme Oxidoreductases
FIGURE 9. Stereo view of the water structure in the active site of NeHAO.
From the central, water-filled cavity (gray surface), two water channels pro-
trude toward the active site (paths 1 and 2). Path 1 could relay protons from
the active site to the central cavity when the conserved histidine is in the
conformation observed in the hydroxylamine soak. In the conformation
observed in the kustc1061-hydrazine soak, path 2 could be used.
6
A, 7D, and 8D), excluding the possibility that the different reac-
tion specificities result from differences in tyrosine binding. Simi-
larly, the direct vicinity of the substrate-binding site on the P
460
co-factor did not immediately explain reaction specificities either.
In as-isolated kustc1061, a water molecule was present as the sixth
(
distal) ligand to the iron in the P co-factor (Fig. 4C). Also in
460
NeHAO, a distal water molecule was bound to the catalytic P
460
co-factor, as deduced from our native (2.2 Å) and recent (2.2 Å)
(
13) resolution structures. This feature could not be discerned
originally because of limited resolution (11).
Substrates Bind to P in Both Kustc1061 and in NeHAO—In
4
60
kustc1061, the water bound to the iron in the P co-factor was
460
replaced by a larger molecule after soaking crystals with
FIGURE 10. View of the active site tyrosine of NeHAO and the active site
methionine of kustc1061 soaked with hydroxylamine. A, stereo view of
hydroxylamine (Figs. 6A, 7A, and 8A). In the resulting 2.2 Å
structure of kustc1061, this compound was modeled as an NO the NeHAO active site as in Fig. 7D, rotated by ϳ90° around the vertical. The
3
58
conserved Tyr provides a possible binding site for a water molecule that
species coordinating at an angle of ϳ107° with respect to the
7
can then attack the proposed {FeNO} species for nitrite formation. B, stereo
heme-iron bond. Considering that hydroxylamine underwent a view of the kustc1061 active site as in Fig. 7A, again after ϳ90° rotation. In
3
23
358
kustc1061, the hydrophobic Met takes the position of Tyr in NeHAO,
precluding the proposed water binding.
two-electron oxidation upon binding, this structure most likely
7
represented a {FeNO} nitrosyl derivative, which was consis-
tent with the observed angle between the NO molecule and the
iron-nitrogen bond. Interestingly, a similarly sized molecule found as well (Figs. 6A, 7D, and 8D). Again, this molecule was
bound with comparable orientation to the P was observed in bound to the heme iron at an angle of 118°, and it was coordi-
460
2
91
292
262
the 1.9 Å electron density maps of a hydrazine-soaked nated by the Asp /His pair, which corresponded to Asp
/
2
63
kustc1061 crystal, possibly representing an iron-bound diazene His
in kustc1061. Probably, the aspartate/histidine pair
(
HN ϭ NH) (Figs. 7B and 8B). Such a diatomic molecule was served to abstract protons from hydroxylamine (Equations 1
not observed in crystals incubated anaerobically with the NO and 2) as was proposed for the orthologous residues in NeHAO
donor 1-(N,N-diethylamine)diazen-1-ium-1,2-diolate. In the (11, 54, 57). Given the flexibility of this region observed in
hydroxylamine-soaked kustc1061 structure, the putative kustc1061 and its proximity to the central, water-filled cavity,
nitrosyl group bound to the P heme iron atom contacted the these residues could shuttle the protons into this cavity, from
460
262
263
conserved aspartate (Asp ) and histidine (His ) residues. where they could be transferred to the solvent. Indeed, in our
The structure of the kustc1061 hydrazine soak was very similar, native NeHAO structure, two chains of water molecules were
262
291
except that Asp had rotated away from the binding site. We observed leading from Asp to the central cavity, which could
also determined the structure of a complex of kustc1061 with a assist in the proposed proton transfer (Fig. 9).
phenyl radical at 2.1 Å resolution by soaking a crystal in phenyl
Ultimately, only one significant structural difference
hydrazine (Figs. 7C and 8C). In this structure, the loop carrying between the kustc1061 and NeHAO active sites was apparent.
262
263
358
the Asp /His pair moved by several Ångstr o¨ ms, which fur- Close to the NeHAO active site, a tyrosine (Tyr ) was present
ther suggested flexibility in this region. In our structure of at the distal side of the porphyrin ring (Fig. 10A). As judged
NeHAO soaked with hydroxylamine, a diatomic molecule was from the amino acid sequence (supplemental Fig. S1), an
JANUARY 17, 2014•VOLUME 289•NUMBER 3
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NO Generation by Octaheme Oxidoreductases
FIGURE 11. Proposed reaction mechanism of hydroxylamine oxidation to NO by kustc1061. The model, based on the NeHAO mechanism (57), shows the
binding of hydroxylamine (H N-OH) to the water-bound resting state (upper left), followed by subsequent oxidation of the substrate and removal of the
2
7
6
protons that proceed through the {FeNO} and {FeNO} nitrosyl states. The model takes into account experimentally established (58, 59) and theoretically
7
predicted (57) reaction intermediates in N-converting ferrihemoproteins, which includes the {FeNO} nitrosyl species observed in the present study.
orthologous tyrosine (Tyr3 ) was present in kustc1061. How-
20
Crystalsbothofkustc1061andNeHAOsoakedwithhydroxyl-
ever, a two-amino acid contraction in kustc1061 resulted in amine showed the presence of a diatomic molecule in the active
320
7
Tyr
being moved away from the active site, where it was site that could be modeled as a {FeNO} nitrosyl (Figs. 6A; 7, A
323
replaced by a hydrophobic methionine (Met ) (Fig. 10B).
and D; and 8, A and D). This would imply that these two proteins
follow a common catalytic cycle (57) at least up to this point,
involving the abstraction of two protons and two electrons (Fig.
DISCUSSION
1
0). Considering that these electrons have to be shuttled one at a
Here, we functionally and structurally characterized the
octaheme protein kustc1061 from the anammox bacterium
K. stuttgartiensis. Despite the limited sequence identity,
kustc1061 shared multiple characteristics with HAO from
N. europaea. Both proteins were organized into covalently
bound homotrimers harboring eight c-type hemes per mono-
mer. The arrangement of these hemes, as well as their compa-
time through the wire of His/His-ligated cytochrome c one-elec-
tron carriers, it is possible to speculate that the tyrosine bound to
the catalytic heme assists in electron transfer by temporarily stor-
ing one of the electrons as a tyrosine radical. The subsequent one-
7
electron oxidation of the {FeNO} nitrosyl would then yield
6
Fe(III)-bound NO ({FeNO} ) as proposed for NeHAO in Ref. 57
(
Fig. 11). Apparently, in kustc1061 this would be followed by the
rable midpoint redox potentials, and the presence of the P
-
4
60
dissociation of NO and the rebinding of water, regenerating the
type catalytic centers were similar. Both kustc1061 and
NeHAO use hydroxylamine as their substrate, but whereas resting state. Although it is known that NO is a minor product of
kustc1061, and presumably many other HAO-like proteins the reaction catalyzed by NeHAO (60), its oxidation reaction has
mentioned below, oxidizes the substrate to NO, nitrite is the to continue to the nitrite state. To reach this state, the NeHAO
(
major) product of NeHAO activity. This resulted in the ques- reaction requires the addition of an oxygen atom, which derived
tion of what determined the reaction specificity.
from a water molecule (57), to the substrate. Comparing the active
1
240 JOURNAL OF BIOLOGICAL CHEMISTRY
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NO Generation by Octaheme Oxidoreductases
sites of kustc1061 and NeHAO, it seems that the reaction specific- REFERENCES
ity relies on facilitating or preventing this step. In NeHAO, the
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26, 3–31
hydrophilic O␩ hydroxyl of the Tyr side chain is perfectly posi-
tioned to bind and orient a water molecule to attack a substrate
bound to the P iron (Fig. 10A). Importantly, in kustc1061 this
2
3
. Hirst, D. G., and Robson, T. (2011) Nitric oxide physiology and pathology.
Methods Mol. Biol. 704, 1–13
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NO signals in the haze. Nitric oxide signalling in plant defence. Curr. Opin.
Plant Biol. 12, 451–458
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particular tyrosine was replaced by the hydrophobic Met (Fig.
1
0B), which would make the binding of a water molecule in this
position unlikely, thus preventing the reaction from proceeding to
4. Wang, Y., and Ruby, E. G. (2011) The roles of NO in microbial symbioses.
Cell Microbiol. 13, 518–526
nitrite.
5
. Lundberg, J. O., and Weitzberg, E. (2013) Biology of nitrogen oxides in the
gastrointestinal tract. Gut 62, 616–629
Hydroxylamine is a potent inhibitor of the N -forming reac-
2
tion catalyzed by the HAO-like hydrazine dehydrogenase
6
. Einsle, O., and Kroneck, P. M. (2004) Structural basis of denitrification.
Biol. Chem. 385, 875–883
(
kustc0694 in K. stuttgartiensis) (8). The presence of kustc1061
provides these microorganisms a means for hydroxylamine
detoxification, at the same time generating NO, a substrate for
hydrazine synthesis (8). Indeed, kustc1061 homologs are found
in all anammox genomes sequenced so far (supplemental Fig.
S1), in which close paralogs may even be observed, such as
kusta0043 in K. stuttgartiensis. Furthermore, hydroxylamine is
a toxic product of ammonium co-metabolism of the particulate
methane monooxygenase, the key enzyme in the highly diverse
an environmentally relevant methanotrophic bacteria. Many of
these organisms harbor at least one HAO-like protein that has
an elusive role. It was suggested that these HAO-like proteins
could either oxidize hydroxylamine to nitrite or NO or alterna-
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