Contrasting Catalytic Profiles of Multi-Heme Nitrite Reductases Containing CxxCK Heme
Binding Motifs.
Rose-Marie A. S. Doyle1, Sophie J. Marritt1, James D. Gwyer1, Thomas G. Lowe1,
Tamara V. Tikhonova2, Vladimir O. Popov2,3, Myles R. Cheesman1 and Julea N. Butt1
1Centre
for Molecular and Structural Biochemistry, School of Chemistry and School of
Biological Sciences, University of East Anglia, Norwich Research Park, Norwich, NR4 7TJ, U.K.
2A.
N. Bakh Institute of Biochemistry RAS, Leninsky pr.33, 119071 Moscow, Russia
3NBICS
Centre of Russian National Research Centre "Kurchatov Institute", Akademika
Kurchatova sq. 1, Moscow 123182, Russia
*Running title: Nitrite Ammonification by Multi-Heme Cytochromes.
 To whom correspondence should be addressed: Julea Butt, School of Chemistry, University
of East Anglia, Norwich Research Park, Norwich, NR4 7TJ, UK, Tel: 00 44 1603 593877, Fax:
00 44 1603 592003, E-mail: j.butt@uea.ac.uk
2
The abbreviations used are: EcNrfA, pentaheme cytochrome with nitrite reductase activity from
Escherichia coli; Em, mid-point potential; LMCT, ligand-to-metal charge-transfer; MCD,
magnetic circular dichroism; MOTTLE, MCD compatible Optically Transparent Thin Layer
Electrochemistry; nIR, near infra-red; PGE, pyrolytic graphite edge; SHE, standard hydrogen
electrode; TvNiR, octaheme nitrite reductase from Thioalkalivibrio nitratireducens, WsNrfA,
pentaheme cytochrome with nitrite reductase activity from Wolinella succinogenes.
Keywords: Magnetic Circular Dichroism, Nitrogen Cycle, Cytochrome, Modified Tyrosine,
Reductive Activation
1
1
2
SUMMARY
3
4
The multi-heme cytochromes from Thioalkalivibrio nitratireducens, TvNiR, and Escherichia coli,
5
EcNrfA, reduce nitrite to ammonium. Both enzymes contain His/His ligated hemes to deliver
6
electrons to their active sites where a Lys-ligated heme has a distal pocket containing a catalytic
7
triad of His, Tyr and Arg residues. Protein film electrochemistry reveals significant differences in
8
the catalytic properties of these enzymes. TvNiR, but not EcNrfA, requires reductive activation.
9
Spectroelectrochemistry implicates reduction of His/His ligated heme(s) as being key to this process
10
which restricts the rate of hydroxide binding to the ferric form of the active site heme. The KM
11
describing nitrite reduction by EcNrfA varies with pH in a sigmoidal manner that is consistent with
12
its modulation by (de-)protonation of a residue with pKa ≈ 7.6. This residue is proposed to be the
13
catalytic His in the distal pocket. By contrast the KM for nitrite reduction by TvNiR decreases
14
approximately linearly with increase of pH such that different features of the mechanism define this
15
parameter for TvNiR. In other regards the catalytic properties of TvNiR and EcNrfA are similar,
16
namely, the pH dependence of Vmax and the nitrite dependence of the catalytic current-potential
17
profiles resolved by cyclic voltammetry, such that the determinants of these properties the enzymes
18
appear to be conserved.
19
2
20
Numerous microbes possess a single enzyme that can reduce nitrite to ammonium without the
21
release of detectable intermediates (1,2). The coordinated delivery of electrons and protons to the
22
substrate ensures that two N-O bonds are cleaved to achieve the overall transformation NO2- + 6e- +
23
8H+ → NH4+ + 2H2O without releasing detectable intermediates. The result is a reaction that
24
removes a potent cytotoxin, serves as a step to assimilate inorganic nitrogen and that may contribute
25
to the generation of a trans-membrane proton gradient. The chemistry that occurs during nitrite
26
ammonification is all the more remarkable when it is recognized that an alternative route to the
27
same transformation employs four enzymes with nitric oxide, nitrous oxide and di-nitrogen as
28
discrete intermediates.
29
One class of nitrite ammonifying enzyme is formed by the multi-heme cytochromes c that
30
contain a CxxCK heme-binding motif (3-10). This motif provides the proximal lysine ligand to the
31
active site heme in addition to cysteine residues that bind the heme through thioether bonds. The
32
sidechains of three conserved residues, namely, His, Tyr and Arg, define the distal pocket of the
33
active site, Fig. 1. Positioned ca. 11 Å from the active site heme iron there is a Ca2+ ion. This
34
contributes to a positive electrostatic potential attracting nitrite to the active site and may play a role
35
in supplying protons to the substrate and catalytic intermediates (11,12).
36
Of the CxxCK containing multi-heme cytochromes c those with five hemes, termed NrfA, are
37
the most extensively characterized. These enzymes contain four low-spin c-hemes, bound through
38
CxxCH heme-binding motifs, which deliver electrons to the active site heme that is high-spin in the
39
absence of substrate or inhibitors (3-8). The low-spin hemes have His/His axial ligation and are
40
positioned as two branches converging on the active site from distinct regions of the protein surface.
41
One branch supports electron exchange between the active site and the cellular redox partner. The
42
second branch allows for electron exchange between NrfA subunits (13) in the homodimeric
43
structures resolved by X-ray crystallography (3-8).
44
With a view to understanding the catalytic mechanism of NrfA several spectroscopic and
45
electrochemical methods have been used with the aim of resolving the redox chemistry of the
46
hemes. There is agreement that the heme reduction potentials span several hundred mV (5,8,14-17).
47
However, there are some differences in the descriptions, Table 1, that are not surprising given the
48
large number of hemes that makes it extremely difficult to unambiguously attribute the
49
contributions from individual centers. X-band electron paramagnetic resonance spectra of the
50
oxidized enzymes are complex and difficult to interpret quantitatively (5,17). The low-spin ferric
51
hemes with perpendicular axial ligand planes, if not spin-coupled, will give rise to resonances of
52
intrinsically low intensity and in addition there is strong spin-coupling between some pairs of
53
adjacent hemes. In contrast, electronic absorbance, magnetic circular dichroism (MCD) and
3
54
resonance Raman spectroscopies are able to unambiguously identify features from all the low-spin
55
His/His ligated hemes and from the high-spin Lysine ligated heme of the active site (15,17).
56
Employing these methods in spectroelectrochemistry, where the sample is equilibrated with the
57
potential applied to an electrode, has resolved redox properties of the NrfA hemes (8,15,17) while
58
avoiding the use of the common reductant sodium dithionite that can generate anions that bind to
59
the active site (18).
60
Cyclic voltammetry can resolve reduction potentials of the NrfA hemes but by itself does not
61
provide structural information to allow their correlation to individual centers (8,15,16). This
62
limitation can be overcome when NrfA is adsorbed as a film on an electrode that allows for
63
spectroscopic characterization of the enzyme in the oxidation state that is defined by the applied
64
electrode potential (15,17). A further and very significant strength of protein-film electrochemistry
65
(PFE) is that it allows the catalytic properties of an enzyme to be resolved as a function of
66
electrochemical potential, time and concentration. As a consequence the information afforded by
67
PFE complements that from other techniques. In addition to defining the kinetic parameters, KM and
68
Vmax, describing steady-state catalysis it is possible to quantify catalytic bias and the rates and
69
mechanisms of inhibition and (in)activation whether reversible or irreversible (19).
70
Largely for the reasons above, PFE has been applied to resolve the catalytic properties of NrfAs
71
from a number of organisms, see for example (13,16,20,21). However, we are not aware that the
72
impact of pH on nitrite ammonification by CxxCK containing multi-heme cytochromes c has
73
received significant attention. Given the importance of protons to nitrite ammonification we address
74
this here in a PFE study that reveals differences in the kinetic parameters that describe the pH
75
dependence of nitrite reduction by Escherichia coli NrfA (EcNrfA) and a closely related enzyme
76
that contains a CxxCK ligated c-heme, namely, Thioalkalivibrio nitratireducens octaheme nitrite
77
reductase (TvNiR) (9). The C-terminal domain of TvNiR contains the constellation of five c-type
78
hemes and axial ligand sets conserved in NrfA. The three additional c-hemes are bound to the N-
79
terminal domain, have His/His axial ligation and extend one branch of hemes leading to the active
80
site. TvNiR catalyzes nitrite ammonification at a lysine ligated c-type heme that, like NrfA, has a
81
distal pocket defined by His, Tyr and Arg side-chains. In a difference to NrfA the Tyr is modified at
82
the ortho-position by a covalent bond to the sulfur of the neighboring cysteine residue, Fig. 1B. To
83
reveal the heme reduction potentials and axial ligand sets that underpin catalysis by TvNiR their
84
magneto-optical and spectroelectrochemical characterization is presented here. These reveal further
85
differences in the properties of the two enzymes.
86
87
Experimental Procedures
4
88
89
Protein Samples and Reagents - The octaheme nitrite reductase from Thioalkalivibrio
90
nitratireducens (TvNiR) was purified according to the published method (22). Purified protein in
91
0.1 M potassium phosphate, pH 7.0 was stored as aliquots in liquid nitrogen. Protein concentration
92
was quantified by electronic absorption spectroscopy using an extinction coefficient of 410 nm = 884
93
000 M-1 cm-1 for air equilibrated (i.e. oxidized) TvNiR. This extinction coefficient was determined
94
following quantification of the c-heme content by the pyridine hemochrome method that was
95
calibrated with horse heart cytochrome c (408 nm = 106 000 M-1 cm-1) (23,24). EcNrfA was purified
96
and characterized as described previously (5). All other reagents were of at least Analar quality and
97
solutions were prepared in water with resistivity > 18 M cm (Purelab Maxima, ELGA).
98
99
Spectroscopic Analysis of Solutions of TvNiR – Samples for ambient temperature MCD of
100
oxidized TvNiR were prepared in 50 mM Hepes, 100 mM NaCl, pH 7.0. Measurements in the near
101
infra-red region and at low temperature were performed on samples prepared in deuterium oxide, 50
102
mM Hepes, pH* 7.0 with 50% glycerol added as glassing agent and where pH* is the apparent pH
103
measured in D2O based solutions using standard glass electrodes. Eu(II) reduced enzyme was
104
prepared by addition of aliquots of 6 mM EuCl2, 50 mM Hepes, pH 7.0 to an anaerobic solution of
105
TvNiR. Eu(II) was added until the electronic absorption spectrum was typical of low-spin ferrous
106
heme and unchanged by further additions of EuCl2 at which point the sample was analyzed by
107
MCD. The aqueous Eu(III/II) couple has Em = -400 mV under the experimental conditions used
108
here (25).
109
Spectroelectrochemical potentiometric titration of TvNiR monitored by MCD used the method of
110
Marritt et al (15) using MCD compatible optically transparent thin layer electrochemistry
111
(MOTTLE). The cell was of 0.5 mm path length and employed a gold mesh working electrode with
112
the following mediators each at 40 µM; ruthenium hexamine chloride, trimethylhydroquinone, 5-
113
hydroxynaphthoquinone,
114
anthraquinone sulfonate, benzyl viologen, sulfonyl viologen and methyl viologen. Spectra from
115
MOTTLE are presented without correction for the zero-field baseline because the time required to
116
cycle the magnetic field of the super-conducting magnet would preclude completion of
117
potentiometric titrations in a practical time period. MCD spectra were recorded on JASCO circular
118
dichrographs, model J810 for the visible region and model J730 for the near infrared region, in a
119
magnetic field provided by an Oxford Instruments superconducting magnet, model SM1 at 6 Tesla
120
for ambient temperature and model SM4 at 5 Tesla for low temperature measurements. It was not
duroquinone,
menadione,
9,10-anthraquinone
2,6
disulfonate,
5
121
possible to access as low potentials in the MOTTLE cell as when studying proteins adsorbed on
122
SnO2 electrodes (15,26). This is most likely due to proton reduction occurring at lower
123
overpotential using the gold-mesh electrodes. All potentials are reported versus the Standard
124
Hydrogen Electrode (SHE).
125
126
Protein Film Electrochemistry – To measure the catalytic activity of TvNiR, films were prepared
127
on freshly polished pyrolytic graphite edge (PGE) electrodes by exposure to 30 µM TvNiR in ice-
128
cold 50 mM Hepes, 100 mM NaCl, pH 7.0 for a few seconds using the procedures previously
129
described for studies of EcNrfA (20). A nitrite stock solution (500 mM) was freshly prepared for
130
each day of experiments and diluted to the required concentration. Cyclic voltammetry and
131
chronoamperometry with rapid rotation of the PGE working electrode were performed using a
132
three-electrode cell configuration housed in a Faraday cage and placed inside a N2-filled chamber as
133
described previously (20). During continuous cyclic voltammetry, and chronoamperometry, the
134
catalytic current magnitudes measured for both EcNrfA and TvNiR showed a first order exponential
135
decay over time. The catalytic current magnitudes were corrected for this effect prior to further
136
analysis such as the extraction of imax and KM values. Catalytic currents were recorded with
137
electrode rotation at 3000 rpm at which speed they were free from limitation by the rate of nitrite
138
delivery to the enzyme film. Kinetic parameters were determined from the variation in catalytic
139
current magnitude (icat) at -550 vs SHE with nitrite concentration ([nitrite]) fitted to the Michaelis-
140
Menten equation:
141
142
nitrite 
i
reaction velocity  icat  max
K M  nitrite 
143
144
where KM is the Michaelis constant and imax is the maximum velocity (Vmax) presented as a catalytic
145
current. The pH dependence of imax (Vmax) was defined by film transfer experiments whereby the
146
catalytic current (ipH) of a given film was measured at the pH of interest, then at pH 7 and then
147
again at the pH of interest with the nitrite concentration in each solution equal to the KM at that pH.
148
Transfer of the film between identical solutions established that the signal loss on film transfer was
149
reproducible but greater than predicted by a first order exponential decay over time. As a
150
consequence, the value of imax relative to that at pH 7 was given by the average of ipHx (initial)/ ipH7 and
151
ipHx (final)/ ipH7. Absolute measurements of imax made with a series of independently prepared films
152
reproduced the variation of imax with pH found in the film transfer experiments. The latter
153
experiment produced a greater scatter of individual data points due to the inherent variation,
6
154
approximately 15%, in current magnitude displayed by independently prepared films measured
155
under identical conditions. This variation is most likely to reflect variation in the population of
156
electroactive molecules in the film forming process. For NrfA the catalytic currents measured above
157
pH 9 were too small for reliable analysis.
158
Spectroelectrochemistry of TvNiR films adsorbed on mesoporous nanocrystalline SnO2
159
electrodes was achieved using minor modification of previously reported procedures (15). Both
160
optical windows of the electrochemical cell supported a SnO2 electrode of geometric area 3 mm × 7
161
mm and 4 μm path length. TvNiR adsorbed onto the electrodes during 4 hours of cyclic
162
voltammetry (+200 to -400 mV) in an anaerobic solution of 15 M TvNiR, 50 mM Hepes, 100 mM
163
NaCl, pH 7.0. Prior to spectroelectrochemistry the electrodes were rinsed thoroughly with 50 mM
164
Hepes, 100 mM NaCl, pH 7.0 to remove non-adsorbed and loosely bound protein.
165
166
Results
167
Magneto-Optical Characterization of TvNiR- Magnetic circular dichroism (MCD) was used to
168
assess the ligation- and spin-states of the hemes in solutions of TvNiR. The air-equilibrated, as
169
prepared enzyme displays a broad trough with a minimum at 620 nm, Fig. 2A. This feature appears
170
in a spectral region where high-spin ferric hemes give rise to ligand-to-metal charge-transfer
171
(LMCT) bands at energies diagnostic of the chemical nature of the axial ligands to the heme (27).
172
For TvNiR the energy of this feature is indicative of a high-spin ferric heme having ligation from a
173
nitrogenous species and hydroxide. The MCD intensity at 620 nm arising from such centers varies
174
from 1 to 4 M-1 cm-1 T-1 per heme (27) and is approximately 2.6 M-1 cm-1 T-1 for TvNiR. This
175
feature is then most reasonably assigned to the active site heme that X-ray crystallography resolves
176
with axial ligation by lysine and an oxygen atom when substrates and inhibitors are absent (9).
177
In the Soret- and /-regions, 400 to 450 nm and 500 to 570 nm respectively, the MCD of
178
TvNiR shows features characteristic of low-spin ferric heme, Fig. 2A. There is no evidence for
179
ferrous-heme. Both low-spin and high-spin ferric heme give rise to a bisignate feature in the Soret
180
region. The peak-to-trough intensity of this feature is significantly greater for low-spin than high-
181
spin heme with typical values being, respectively, 150 M-1 cm-1 T-1 and 20 M-1 cm-1 T-1 per heme
182
(28). For TvNiR the intensity of this feature is approximately 1100 M-1 cm-1 T-1 and so is consistent
183
with a nearest integer ratio of seven low-spin ferric hemes per high-spin heme. Low-spin ferric
184
hemes display LMCT bands in the near infra-red region with energies that reflect the chemical
185
nature of their axial ligands (29). For TvNiR the single positive band at 1500 nm with a vibrational
7
186
sideband at shorter wavelengths (1200 to 1300 nm) is typical for His/His ligated ferric heme, Fig.
187
2B. The MCD provides no evidence for low-spin hemes with other ligand sets.
188
To assess the nature of the hemes present in reduced TvNiR the enzyme was reduced with
189
aqueous Eu(II). Aliquots of Eu(II) were added until the electronic absorbance was unchanged by
190
further additions and typical of that from low-spin ferrous c-heme with maxima at 424, 524.5 and
191
554 nm, Fig. 2C inset. The ambient temperature MCD of the Eu(II) reduced TvNiR in the visible
192
and Soret regions showed features that could be confidently assigned to low-spin, but not high-spin,
193
ferrous heme (not shown). This could indicate an absence of high-spin ferrous heme or simply the
194
difficulty of resolving features characteristic of high-spin ferrous heme in the presence of the much
195
more intense features that arise from low-spin ferrous heme. To resolve these possibilities MCD
196
was performed at temperatures ≤ 100 K, Fig. 2C.
197
The intensities of features from paramagnetic high-spin (S=2) states ≤ 100 K are strongly
198
temperature dependent whereas those from diamagnetic low-spin (S=0) states are temperature
199
independent (30). At the relatively high temperature of 100 K, the MCD of TvNiR that has been
200
reduced by aqueous Eu(II) is dominated by sharp bisignate features between 500 and 560 nm that
201
are typical of low-spin ferrous heme, Fig. 2C. These features have the same intensities at 100, 10
202
and 4.2 K since they arise from diamagnetic centers. By contrast the intensities of the features in the
203
Soret region increase significantly as the temperature is lowered and must arise from paramagnets;
204
the 440 nm peak and 427 nm trough are typical of high-spin ferrous heme. The only exception to
205
this is a small positive and temperature invariant feature at ≈ 422 nm that is the Soret band of the
206
diamagnetic low-spin hemes. The 397 nm peak and 410/416 nm trough are characteristic of low-
207
spin ferric heme (S = ½).
208
The absolute intensities of the MCD features at each temperature ≤ 100 K indicate that addition
209
of Eu(II) has achieved greater than 85% reduction of TvNiR (30). A single high-spin ferrous heme,
210
most likely to be that associated with the active site, is detected together with at least six low-spin
211
ferrous hemes. The MCD of air equilibrated and Eu(II) reduced TvNiR is consistent with the hemes
212
having the same axial ligation as resolved by X-ray diffraction where the oxidation state of the
213
enzyme is unknown.
214
215
Spectroelectrochemical Characterization of TvNiR –Initial analysis of TvNiR redox chemistry was
216
performed following adsorption of the enzyme onto optically transparent, mesoporous,
217
nanocrystalline SnO2 electrodes. With the electrode poised at potentials above +50 mV the
218
electronic absorbance spectra contained a Soret maximum at 410 nm alongside weaker features
219
between 500 and 600 nm, Fig. 3A. These features are typical of the oxidized enzyme, e.g., Fig. 2C,
8
220
and here they are superimposed on a slope due to scattering by the SnO2 particles. Assuming the
221
extinction coefficient of the adsorbed protein to be that of the protein in solution, and given that the
222
actual area of the electrode is 300 the geometric area (31), the amount of adsorbed TvNiR was
223
calculated as approximately 1 picomole cm-2 of the actual area.
224
When the electrode was poised below -500 mV the electronic absorbance failed to resolve
225
features of the oxidized protein. Instead maxima at 423, 524 and 553 nm were displayed. These are
226
characteristic of reduced TvNiR and demonstrated that all the adsorbed protein was able to
227
equilibrate with the potential applied to the electrode, Fig. 3A. The non-turnover peaks resolved by
228
cyclic voltammetry of the adsorbed protein were not sufficiently defined to be confidently resolved
229
from the background of much larger and non-uniform currents arising from charging of the
230
nanostructured SnO2 (31,32). Nevertheless, unambiguous and background free detection of the
231
redox changes in adsorbed TvNiR was afforded by the potential dependence of its electronic
232
absorbance. This was independent of whether the potentiometry was performed in an oxidative or
233
reductive direction, Fig. 3A, inset.
234
The ferric/ferrous couple of low-spin, but not high-spin, c-heme contributes to the change in
235
absorbance at 553 nm. This is observed here between +160 and -450 mV and so across a similarly
236
low potential window to that where the NrfA low-spin hemes are redox active, Table 1 (8,14,15).
237
This data is not readily resolved into the individual contributions of the seven His/His ligated hemes
238
in TvNiR. However, an indication of their macroscopic reduction potentials, Em, can be presented if
239
several assumptions are made, namely, that each heme contributes equally to the total absorbance
240
change and acts independently in a reversible manner described by the Nernst equation for a one-
241
electron (n = 1) process. With these assumptions the least number of components required to
242
describe the plot of 553 nm absorbance versus potential is four with Em values of +100 (one heme),
243
-80 (one heme), -200 (three hemes) and -370 (two hemes) mV, Fig. 3A inset. Within our model an
244
uncertainty of 20 mV is associated with each Em to reflect the accuracy with which the absolute
245
value of our reference electrode is known.
246
In favorable circumstances the ferric/ferrous transformation of high-spin heme can be detected
247
through changes in the absorbance around 440 nm, e.g. (15,33). Such changes are most readily
248
identified in reduced minus oxidized difference spectra generated for closely spaced equilibration
249
potentials since the redox transformation of low-spin hemes can also give rise to spectral changes at
250
this wavelength. However, for TvNiR the difference spectra are dominated by features typical of the
251
ferric/ferrous transition of low-spin c-hemes with no feature(s) that could be assigned to the
252
corresponding transition of a high-spin heme, Fig. 3B. This is most likely because the absorbance
253
change per low-spin heme is intrinsically of much larger magnitude than the absorbance change per
9
254
high-spin heme, a situation that is compounded in TvNiR by the presence of seven low-spin hemes
255
for each high-spin center. As a consequence evidence for redox transformation of the high-spin
256
heme was sought from MCD-monitored spectroelectrochemistry where the 620 nm trough
257
measured at ambient temperatures reports on the population of high-spin ferric heme with
258
lysine/hydroxide ligation.
259
The intrinsically low intensity of the 620 nm feature required spectroelectrochemistry of highly
260
concentrated TvNiR solutions. For this an optically transparent thin-layer electrochemical cell
261
employing a gold-mesh working electrode was used together with a cocktail of redox mediators as
262
described in the Experimental Procedures. The spectral features arising from both the ferric and
263
ferrous states of the low-spin hemes displayed fully reversible behavior consistent with that
264
displayed by the enzyme adsorbed on SnO2 electrodes (not shown) and equilibration with the
265
applied potential was typically achieved within 40 minutes. The active site heme behaved
266
differently in that re-oxidation occurred over a much longer time frame than reduction. The
267
intensity of the 620 nm trough decreased to a negligible level as the potential of the sample was
268
lowered from -200 mV to -275 mV, Fig. 4A. However, even after 17 hrs exposure to +180 mV the
269
intensity of this feature was not restored to that displayed by the protein as purified. After 30
270
minutes the intensity was approximately 40% of that observed initially and a second trough, not
271
present in the spectrum of the as-prepared enzyme, was apparent at 630 nm, Fig. 4B. After 17 hours
272
the 620 nm trough had been restored to 60% of its original intensity and there was no indication of
273
additional negative intensity at longer wavelengths. The transient appearance of a trough at 630 nm
274
on re-oxidation of TvNiR suggests a Lys/water ligated high-spin ferric heme (34) as an intermediate
275
in the pathway of oxidizing the high-spin ferrous heme to its thermodynamically most stable
276
Lys/hydroxide ligated ferric state.
277
278
TvNiR nitrite reductase activity resolved by protein film electrochemistry and compared to that of
279
EcNrfA – PFE was used to resolve the catalytic activity of TvNiR and EcNrfA as a function of pH.
280
For this the mesoporous nanocrystalline SnO2 electrodes were unsuitable. Substrate mass transport
281
to enzyme molecules adsorbed deep within the mesoporous structure limits the catalytic current and
282
masks the intrinsic properties of the enzyme (35). Instead both enzymes were adsorbed on PGE
283
electrodes that were rotated rapidly to further facilitate substrate delivery to the electrode surface.
284
Cyclic voltammetry of freshly polished PGE electrodes exposed to a few microlitres of TvNiR,
285
placed in 0.1 mM nitrite and rotated at 3000 rpm gave rise to negatively signed, and so reductive,
286
catalytic currents below -100 mV, Fig. 5A. The catalytic response did not change on addition of 100
287
mM phosphate but it was lost on introducing 500 µM cyanide, not shown. (Note: cyanide is toxic
10
288
and extreme care should be taken in its handling and disposal.) These observations correlate with
289
the catalytic properties of TvNiR resolved by spectrophotometric assays using dithionite reduced
290
methyl viologen as electron donor (22). It was concluded that TvNiR, like EcNrfA (20,36), adsorbs
291
on PGE electrodes in an electrocatalytically active form that retains the characteristic properties of
292
the enzyme in solution. Non-turnover peaks whose area would quantify the amount of electroactive
293
enzyme were not visible for either enzyme and this is most likely related to poor surface coverage.
294
This includes the possibility of a distribution of orientations on the surface many of which are
295
incapable of direct electron exchange with the electrode and it prevents the calculation of turnover
296
numbers for nitrite reduction. Nevertheless the variation in catalytic current with electrochemical
297
potential, time and pH provides much insight into the catalytic performance of the enzymes as
298
described below.
299
The catalytic properties of TvNiR are more apparent when the voltammetric response of the bare
300
electrode is subtracted from that displayed by the enzyme film under otherwise identical conditions,
301
Fig. 5B. The catalytic current magnitudes of the first reductive scan from +200 to -600 mV, Fig. 5B
302
bold continuous line, are smaller than those resolved on returning to more positive potentials, Fig.
303
5B thin continuous line. The catalytic current-potential profiles of the subsequent cyclic
304
voltammograms do not show this hysteresis, Fig. 5B broken lines. The profiles for cyclic
305
voltammograms 2 to 5 are independent of scan direction aside from a first order loss of catalytic
306
current magnitude over time that is typical of such experiments and attributed to loss of
307
electrocatalytically active enzyme. Significantly the catalytic current-potential profiles of scans 2 to
308
5 resemble that seen on returning to positive potentials during the first voltammogram. Taken
309
together the cyclic voltammetry describes an irreversible reductive activation, on the time scale of
310
the PFE experiment, of the TvNiR film on scanning from +200 to -600 mV for the first time. Such
311
behavior has not been noted for EcNrfA. The irreversible reductive activation is distinct from the
312
reversible modulation of activity that is observed on subsequent cycling of the enzyme from more
313
positive to more negative potentials. The latter is expected as the enzyme is swept between the
314
oxidized, catalytically inactive, and reduced, catalytically active, oxidation states.
315
Chronoamperometry was employed to resolve the rate of irreversible reductive activation as a
316
function of electrochemical potential, pH and nitrite concentration. This has the advantage of
317
avoiding the intimate linkage of time and electrochemical potential that is intrinsic to cyclic
318
voltammetry. The electrode potential was stepped between defined values and the subsequent
319
relaxation of catalytic current reported on the rate of any reductive activation that occurred. A
320
typical result is illustrated in Fig. 6A. Initiating the experiment at +140 mV with the electrode
321
rotating rapidly in 0.1 mM nitrite there is little flow of current as expected from cyclic voltammetry,
11
322
Fig. 5. The small drop of current noted in the first 20 seconds arises from charging of the electrical
323
double-layer. On stepping the potential to -160 mV there is an immediate and significant increase in
324
the catalytic current as anticipated from cyclic voltammetry, e.g. Fig. 5. The current due to double-
325
layer charging decays over 10 seconds after which time a constant catalytic current is measured.
326
Stepping the potential to -270 mV elicits an immediate increase in catalytic current, again as
327
expected from the cyclic voltammetry, followed by a further significant increase in the catalytic
328
current over 120 seconds that describes the reductive activation of the protein film. Returning the
329
electrode potential to -160 mV shows the catalytic current had increased by approximately 30%
330
from that prior to reductive activation. Equilibrating the enzyme at -310 mV and returning to -160
331
mV provided no evidence for further activation of the film.
332
In the experiment described above the reductive activation of TvNiR at -270 mV is well-
333
described as a first order process with a rate constant of 0.030 ± 0.005 s -1, Fig. 6A circles.
334
Reductive activation was studied in a similar manner for a number of potentials in the pH range 6 to
335
10. Regardless of conditions, complete activation of the film produced a 30% increase of activity.
336
Reductive activation was too fast to quantify when the rate constant exceeded 0.7 s-1 but the data
337
clearly demonstrated how for a given potential the reductive activation was faster at more acidic
338
pH, Fig. 6B. Further experiments revealed that neither the rate nor the extent of reductive activation
339
were dependent on nitrite concentration (not shown). When considering the possible rate-defining
340
event in reductive activation the process of intramolecular electron transfer seems an unlikely
341
candidate due to the proximity of adjacent hemes within TvNiR (37). A simple description of the
342
reductive activation then involves interfacial electron transfer to an activating center followed by a
343
chemical event that produces the more active enzyme. In this model two limiting behaviors can be
344
put forward to account for the variation of reductive activation rate constant with electrochemical
345
potential. When the rate is defined by slow interfacial electron transfer the rate constant will
346
increase exponentially with overpotential as described by the Butler-Volmer equation (38). This
347
behavior is illustrated by the red line in Fig. 6B that has been generated using parameters, stated in
348
the figure legend, that produce a reasonable correlation with the data at pH 7. However, the
349
correlation with the data is not strong and the catalytic currents supported by TvNiR suggest
350
interfacial electron transfer is not rate-limiting. Taking 0.05 picomoles TvNiR as an estimate of the
351
upper limit of electroactive coverage based on failure to resolve non-turnover peaks when nitrite is
352
absent indicates that each molecule of adsorbed TvNiR supports a minimum electron flux of 500 s-1.
353
This is significantly greater than the rate constants for reductive activation and consistent with
354
solution assays that report an electron flux through TvNiR of the order of 18 000 s-1 (10).
12
355
In the absence of rate-defining interfacial electron transfer the rate of reductive activation will be
356
defined by the chemical step that follows reduction of the activating center. The rate constant will
357
reflect the population of enzyme with the activating center reduced and vary with electrochemical
358
potential in a sigmoidal manner as predicted by the Nernst equation and centered on the mid-point
359
potential of the activating center (39). This is illustrated by the solid black line in Fig. 6B that show
360
best fit to the pH 7 data for an n =1 center. The lack of data at low potentials requires that the
361
precise values of the parameters producing this fit are interpreted with caution but the model better
362
reproduces the pH 7 data than the prediction from rate-limiting interfacial electron transfer. The pH
363
dependence of the plots of rate constant versus electrochemical potential indicates a positive shift of
364
Em for the activating center with decrease of pH and so a reduction that is accompanied by
365
protonation.
366
To define the KM and maximum catalytic current (imax, that is equivalent to Vmax) describing
367
steady-state nitrite reduction by TvNiR, nitrite was titrated into the electrochemical cell after two
368
cyclic voltammograms had been measured to ensure complete activation of the enzyme film, e.g.
369
Fig. 5 and 7. Parallel experiments were performed in solutions having a pH value between 5 and 10.
370
Visual inspection of the voltammograms presented in Fig. 7 readily illustrates how lower values of
371
imax and KM are displayed at more positive potentials. In this work analysis was performed at -550
372
mV to facilitate comparison of the catalytic properties of the adsorbed enzymes with those
373
displayed in solution phase spectrophotometric assays with methyl viologen (Em,7 -550 mV) as
374
electron donor (5,10). At each pH the variation of catalytic current magnitude with nitrite
375
concentration was well-described by the Michaelis-Menten equation, e.g., Fig 6 inset.
376
Nitrite reduction by TvNiR at pH 7 was described by a KM of 223 ± 60 µM that is in good
377
agreement with the value of 180 ± 50 µM determined by spectrophotometric assay (10). The value
378
of imax decreased in an approximately linear fashion, Fig. 8A, as the pH increased while that of KM
379
decreased 10-fold in an approximately linear manner, Fig. 8B. Neither behavior is readily explained
380
by the sigmoidal variation with pH that is expected when the protonation state of a single ionizable
381
residue modulates these parameters. A series of parallel experiments established that the nitrite
382
reductase activity of EcNrfA follows Michaelis-Menten kinetics over this pH range (20). The pH
383
dependence of the maximum activity of EcNrfA is very similar to that displayed by TvNiR, Fig. 8A.
384
However, the pH dependence of KM is different. For EcNrfA the pH dependence of KM is well
385
described by the behavior expected for ionization of a single residue with pKa = 7.6 ± 0.3 where the
386
protonated residue results in lower values of KM.
387
388
13
389
Discussion.
390
Nitrite ammonification is a complex process and one for which the molecular details of the
391
catalytic cycle remain to be fully understood. In this context it is striking that TvNiR and EcNrfA
392
differ in aspects of their catalytic behavior despite their structural similarities. That His, Tyr and
393
Arg are conserved in the distal pocket of the active sites of TvNiR, EcNrfA and their homologs
394
indicates a critical role for each of these residues in catalysis. X-ray crystallography provides
395
evidence for the residues that are important in binding nitrite. The structure of EcNrfA is very
396
similar to that of Wolinella succinogenes NrfA (WsNrfA) and the latter enzyme has been co-
397
crystallized with nitrite (40). One nitrite oxygen lies within H-bonding distance of the N atom of
398
His and the second oxygen within H-bonding distance of the N2 atom of Arg. The Tyr OH is
399
outside H-bonding distance of the closest nitrite oxygen and density functional theory supports key
400
roles for His and Arg, but not Tyr, in nitrite binding (11). A pKa ≈ 8.2 was previously assigned to
401
the active site His in Desulfovibrio desulfuricans NrfA (16). If the equivalent His in EcNrfA is
402
deprotonated with a pKa  7.6 this can account for the pH dependence of this enzyme’s KM that can
403
be simply considered to reflect the affinity of the active site for nitrite. His deprotonation will
404
remove an opportunity for H-bonding and lower the positive charge in the active site. Both effects
405
will lower the affinity for nitrite and increase the KM describing nitrite reduction by EcNrfA. Such a
406
model does not account for the pH dependence of the KM describing TvNiR nitrite reduction.
407
The active site of TvNiR is smaller than that of EcNrfA and it binds nitrite through H-bonds from
408
His and Tyr OH to one oxygen and from Arg to the second oxygen (9). This may result in
409
differences in the absolute affinities of TvNiR and EcNrfA for nitrite. However, it does not readily
410
explain why the KM for TvNiR nitrite reduction increases as the pH drops. Within the active site of
411
TvNiR the His is expected to contribute to catalysis in much the same way as the equivalent residue
412
in EcNrfA. That contribution to the mechanism must be overridden in terms of defining the KM for
413
TvNiR nitrite reduction if we accept that His (de-)protonation defines the pH dependence of the KM
414
displayed by EcNrfA. In TvNiR the KM must instead have defining contributions from rates
415
associated with the transformation of catalytic intermediates and/or the microscopic steps in
416
substrate delivery to the active site). Differences in the structures of the two enzymes point to the
417
features that will be responsible for this. One is the covalent modification by Cys of the active site
418
Tyr in TvNiR (9). This will lower the Tyr reduction potential by up to 0.4 V but as yet there is no
419
evidence that a Tyr radical contributes to catalysis (4,12). Since replacing Tyr with Phe produces an
420
enzyme unable to reduce nitrite to ammonium (41) a key role for this residue in supplying protons,
421
directing nitrite into the active site or releasing reaction products must then be inferred. In each of
14
422
these functions lowering of the Tyr pKa by up to 1 unit on covalent modification may account for
423
their different rates within EcNrfA and TvNiR and, as a consequence, the different pH dependences
424
of their KM values.
425
There are further differences between the two enzymes when the details of the two channels
426
linking their deeply buried active sites to the bulk solution are considered. These will no doubt
427
contribute to different rates for the microscopic steps in delivering nitrite to the active site but
428
whose calculation is beyond the scope of this study. However, one feature of these channels does
429
warrant comment. In EcNrfA a positively charged channel directs substrate to the active site and a
430
negatively charged channel allows for product egress. In TvNiR it is striking that both channels
431
have positive charge at the protein surface. This is reminiscent of the charge distribution in the
432
nitrite channel NirC that spans the inner membrane of Salmonella typhimurium (42) such that nitrite
433
may have opportunity to explore the two channels of TvNiR than just a single channel of EcNrfA.
434
The catalytic properties of TvNiR and EcNrfA are further distinguished by the reductive
435
activation displayed by the former enzyme. While the active site of TvNiR is purified with
436
His/hydroxide ligation it is unlikely that this species appears in the catalytic cycle since it is formed
437
over a time-scale of hours following reduction and reoxidation of the enzyme. The high-spin ferrous
438
heme in TvNiR is most likely to be penta-coordinate (43) and it may well re-oxidise in this state
439
such that penta-coordinate species key to catalysis. A penta-coordinate, high-spin heme with Lys
440
ligation would have a LMCT band at 660 nm. Such features have significantly lower intensity than
441
those from the hexa-coordinate counterparts (see (44) and references therein) and as a consequence
442
it is not surprising that it goes undetected in our experiments. However, this hypothesis suggests
443
that reductive activation is achieved by preventing hydroxide ligation of the active site heme in its
444
ferric state. This is not a direct effect since reduction of the Lys/hydroxide ligated heme occurs at
445
potentials that are more positive than those required to see reductive activation, Fig. 6B. Rather
446
reduction of low-spin heme(s) is responsible for this effect. The low-spin heme adjacent to the
447
active site heme is axially coordinated by the His that is two residues from the Tyr of the distal
448
pocket, Fig. 9. Movement of this His on reduction of the low-spin heme may alter the disposition of
449
residues in the active site distal pocket to slow the rate of hydroxide binding.
450
Given the discussion above it is striking that in other regards the catalytic properties of TvNiR
451
and EcNrfA are so similar, namely, the pH dependence of Vmax and the nitrite dependence of the
452
catalytic current-potential profiles resolved by cyclic voltammetry. The determinants of these
453
properties the enzymes appear to be conserved. Detailed discussion of the possible origin of the
454
catalytic current-potential profiles has been presented previously for EcNrfA (13,19,20,45). The pH
455
dependence of Vmax may reflect the rate of supplying protons to the active site and/or the roles of
15
456
multiple residues in delivering the eight protons that are required to transform nitrite to ammonium.
457
In the future it is hoped that studies of the pH dependence of the transformation of nitric oxide and
458
hydroxylamine, proposed intermediates in nitrite reduction, together with engineered variants of
459
both enzymes will shed greater insight into the molecular origins of each of the properties described
460
here. Such studies may also help to elucidate the degree to which the catalytic properties of these
461
enzymes arise from the environments colonized by the microbes from which they originate. Both
462
enzymes are periplasmic so they will experience the pH of these environments. For TvNiR this will
463
be pH 10 of the hypersaline alkaline lakes of Wadi Natrun (Egypt) (46). For EcNrfA this will range
464
from pH 2 to 9 during colonization of the gastrointestinal tract. This may have resulted in EcNrfA
465
developing the ability to scavenge, and reduce, nitrite at low pH a feature not needed by TvNiR.
466
In closing we note that the results presented here for TvNiR illustrate the difficulties that can be
467
experienced in using (magneto-)optical methods to resolve redox properties of a high-spin c-heme
468
when low-spin c-hemes are present. It has already been mentioned that the signals from the former
469
are of intrinsically lower intensity than those from the later. Nevertheless it should be possible to
470
resolve the Em of the high-spin heme if it is sufficiently separated from those of the low-spin hemes
471
as for EcNrfA (15). In PFE the potential of the onset of catalysis detected during cyclic
472
voltammetry at low substrate concentration is often directly related to the active site reduction
473
potential (19). On this basis the Em of the catalytically relevant ferric/ferrous couple of the TvNiR
474
high-spin active site heme is likely to lie a little below -0.1 V, in the potential window spanned by
475
redox transformation of its low-spin hemes, and so to have a value similar to that of the active site
476
heme in EcNrfA. This is consistent with reduction of the active site occurring only when other
477
hemes are reduced such that nitrite binding may be prevented until sufficient electrons are present
478
to avoid release of the as yet undetected intermediates in nitrite ammonification. The Em values of
479
the active sites in both enzymes would also then be consistent with their ability to reduce nitrite to
480
ammonium and overcoming the reduction potentials associated with the proposed intermediates in
481
the reaction pathway; nitrite/nitric oxide (ca. +0.18 V), nitric oxide/hydroxylamine (ca. -0.06 V)
482
and hydroxylamine/ammonium (ca. +0.87 V). By contrast potentials a little below +0.3 V are
483
sufficient to detect nitrite reduction to form nitric oxide by the copper-containing enzyme from
484
Alcaligenes faecalis (47).
485
486
Acknowledgements - We are grateful to Professor James Durrant and Li Xiaoe (Imperial College
487
London) for providing the SnO2 electrodes used in this work and to the reviewers for their insights
488
into the interpretation of the data presented in the manuscript. The work was funded by the UK
16
489
Biotechnology and Biosciences Research Council through grants B15211, C007808, B18695,
490
G024758 and G009228.
491
492
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19
Table 1 Summary of Em values for TvNiR obtained in this work and for NrfA from a number of organisms
obtained in previous studies.
Protein
Method
Em (mV) vs SHE
Escherichia coli
Spectroelectrochemistry
-20 (lsa), -108 (hsa), -153 (ls), -206 (ls), -292 (ls)
NrfA (15)
(MCD)
PFE
+22 (ls), -56 (hs), -117 (hs), -189 (hs), -275 (ls)
(SnO2 electrode)
Shewanella
Spectroelectrochemistry
oneidensis MR1
(electronic absorbance)
NrfA (8)
PFE
-62, -140, -250, -283, -342
-36, -105, -166, -230, -295
(graphite electrode)
Desulfovibrio
Spectroelectrochemistry
vulgaris NrfHA
(resonance Raman)
-50 (hs)
(17)
Desulfovibrio
Potentiometry b
desulfuricans
(EPR and Mössbauer)
NrfHA (14,16)
PFE
+150 (ls), -50 (ls), -80 (hs), -480 (ls, 2 hemes)
-220 (active site CO binding heme)
(graphite electrode)
Thioalkalivibrio
Spectroelectrochemistry
nitratireducens
(electronic absorbance, MCD)
+100 (ls), -80 (ls), -200 (3, ls) -370 (2, ls)
TvNiR
(this work)
a
ls low-spin, hs high-spin
b
Dithionite or Zn as reductant
20
Fig. 1. The active sites of two nitrite ammonifying enzymes that contain CxxCK coordinated
active site hemes Ec NrfA (A) and TvNiR (B).
The catalytic triad of Tyr, His and Arg in the distal pocket are illustrated together with the Cys
modification of the active site Tyr in TvNiR. In Ec NrfA Phe218 (blue) is the equivalent residue to
Cys305 that forms a covalent link to Tyr303 in TvNiR. The Ca2+ located adjacent to the active site
is shown as a magenta sphere.
Fig. 2. Spectroscopic properties of TvNiR.
(A) Ambient-temperature MCD of oxidized TvNiR at 8 µM for 300 to 450 nm and 500 M for 450
to 750 nm. Samples in 50 mM Hepes, 100 mM NaCl, pH 7.0. (B) Low-temperature MCD of 70 µM
oxidized TvNiR at near infra-red wavelengths measured at 4.2 K and 5 T in 25 mM Hepes, 50%
glycerol D2O, pH* 7. (C) Low-temperature MCD of TvNiR reduced by EuCl2. Spectra were
measured at 5 T with the sample in 25 mM Hepes, 50% glycerol, pH 7. Inset, electronic absorbance
of oxidized TvNiR and the Eu(II) reduced enzyme prior to freezing for MCD; y-axis ε (mM-1 cm-1).
TvNiR was 11.6 M.
Fig. 3. Potentiometric titration of TvNiR adsorbed on mesoporous nanocrystalline SnO2
monitored by electronic absorption spectroscopy.
(A) Electronic absorption spectra for the enzyme equilibrated at +0.3, -0.115, -0.215, -0.315 and 0.515 V. The arrows indicate the direction of spectral changes on application of increasingly
negative potentials. Inset: Extent of reduction determined from ∆A553
nm
for reduction (●), re-
oxidation (□), and re-reduction (○) where the line shows the sum of seven Nernstian contributions
of equal magnitude with Em= 0.1 (one heme), -0.08 (one heme), -0.20 (three hemes), -0.37 (two
hemes) V, see text for details.
(B) Difference spectra for the indicated potentials. The vertical bar corresponds to an absorbance
difference (A) = 0.1. Buffer electrolyte was 50 mM Hepes, 100 mM NaCl, pH7.
Fig. 4. Potentiometric titration of TvNiR monitored by ambient temperature MCD.
(A) MCD of TvNiR equilibrated at increasingly negative potentials of +0.15, -0.115, -0.215, -0.245
and -0.275 V as indicated by arrow. Inset: normalized variation of MCD intensity, 620 nm minus
634 nm, with equilibration potential in Volts and a line to guide the eye that is centered on -230
mV. (B) MCD of TvNiR as isolated (light solid line) and following reduction held at +180 mV for
21
30 minutes (heavy solid line) and 17 hrs (broken line). TvNiR (500 M) in 50 mM Hepes, 100 mM
NaCl, pH 7 with a mediator cocktail as described in the Experimental Procedures.
Fig. 5. Cyclic voltammetry of a freshly prepared TvNiR film in 0.1 mM nitrite, pH 7.0.
(A) Continuous lines show the initial (bold) and four subsequent cyclic voltammograms from a
TvNiR film. Broken lines show the voltammograms from an equivalent experiment in the absence
of TvNiR. The arrows indicate the direction of scanning. Cyclic voltammetry was at 30 mV s -1 with
electrode rotation at 3000 rpm in 20 mM Hepes, 100 mM NaCl, 20oC. (B) The catalytic currentpotential profiles of TvNiR obtained from the data in (A) as the difference between the currents
recorded in the presence and absence of TvNiR. Bold lines are for the first cyclic voltammogram,
arrows indicate scan direction. Cross-hairs indicate the zero of current and potential.
Fig. 6. Reductive activation of TvNiR resolved by chronoamperometry.
(A) Chronoamperometry of a TvNiR film in 0.1 mM nitrite, pH 7.0. The potential applied to the
electrode for each 120 sec period is indicated. Circles show the fit to a first order increase with a
rate constant of 0.03 s-1. The catalytic current was derived from the raw data by subtraction of the
current recorded in an identical experiment performed in the absence of TvNiR followed by first
order compensation to account for the loss of signal over time. (B) First order rate constants (s-1)for
the reductive activation of TvNiR at pH 10 (star), 9 (closed circle), 8 (square), 7 (triangle), and 6
(open circle). The black lines show the concentration of reduced species predicted by the Nernst
equation for an n =1 couple with Em = -0.42 (pH 8), -0.36 (pH 7) and -0.27 (pH 6) V. The blue line
shows the fraction of reduced active site heme predicted by reductive titration of the 630 nm MCD
trough, Fig.4A, normalized to 1.0. The red-line illustrates the heterogeneous rate constant (cm s-1)
predicted by the Butler-Volmer equation for an n=1 process with Em = -0.28 V and ko = 0.03 cm s-1.
Experimental conditions as in Fig. 2.
Fig. 7. The steady-state nitrite reductase activity of TvNiR at pH 7.0.
(A) Steady-state catalytic currents recorded from a single film of TvNiR are shown for the nitrite
concentrations indicated. Cyclic voltammetry was at 30 mV s-1 with electrode rotation at 3000 rpm
in 20 mM Hepes, 100 mM NaCl, 20 oC. Inset: Michaelis-Menten plot derived from the steady-state
catalytic currents at -0.55 V where the line shows the best fit to the data with KM = 0.237 mM and
imax (Vmax) = 8 µA, see text for details.
22
Fig. 8. pH dependence of the kinetic parameters describing steady-state nitrite reduction by
TvNiR (open circles) and E. coli NrfA (closed circles).
The line in panel B describes the variation in KM for NrfA through the ionization of a single residue
with a pKa = 7.6. Buffer-electrolyte was 100 mM NaCl with 20 mM each of Hepes, Taps, Mes and
acetate for TvNiR and 2 mM CaCl2 with 25 mM each of Hepes, Mes, Taps and acetate for E. coli
NrfA. All experiments at 20 oC.
Fig. 9. The active site heme (red) and adjacent His/His ligated heme (green) in TvNiR. Two
residues separate the catalytic Tyr in the distal pocket of the active site from a His ligand of the
adjacent heme.
23
A
B
Fig. 1.
24
-1
-1
-1
/H (M .cm .T )
A
300
0
x70
-300
x10
-600
2000
500
600
700
B
-1
-1
 (M .cm )
400
1000
0
1000
-1
5
 (mM .cm )
10
-1
15
1500
2000
440 nm
C
1000
397 nm
500
0
0
400
600
-5
B K
4.2
J K
10
F K
100
-10 410 nm
416 nm
427 nm
-15
400
500
600
700
Wavelength (nm)
Fig. 2.
25
A
100
Reduction (%)
Absorbance
1.2
0.8
0
0.4
-0.4
0.0
0.4
Potential (V)
0.0
Absorbance Difference
B
-0.115 - (0.30)V
-0.165 - (0.115)V
-0.215 - (-0.165)V
-0.265 - (-0.215)V
-0.365 - (-0.315)V
-0.415 - (-0.365)V
-0.465 - (-0.415)V
-0.515 - (-0.465)V
400
500
600
700
Wavelength (nm)
Fig. 3
26
A
-2
100
% Reduction
∆/H (M-1 cm -1 T-1)
0
-4
∆/H (M-1 cm -1 T-1)
600
620
0
-0.4
640
0.0 (V)
660
B
20
0
-20
7
-40
500
600
Wavelength (nm)
Fig. 4.
27
A
Scan
5
++
Start
1
Scan
5
1
++
B
Scan
5
1 µA
1
-0.6
-0.3
0.0
Potential (V) vs S.H.E.
Fig. 5.
28
A
Catalytic Current (µA)
0.0
+0.14
-0.16
-0.27
-0.16
-0.31
-0.16
Electrode
Potential
(V)
-2.0
-4.0
0
120 240 360 480 600 720
Time (sec)
B
1
1
Rate Constant
[Reduced] (norrmalized)
0
-0.5
-0.4
-0.3
-0.2
0
-0.1
Electrode Potential (V) vs SHE
Fig. 6.
29
0.0
[nitrite]
mM
-2.0
0.08
0.16
0.32
-4.0
1.28
2.56
10
Catalytic Current
Magnitude (A)
Catalytic Current (µA)
0.01
0
0 [nitrite] (mM) 6
-0.6
-0.3
0.0
Potential (V) vs S.H.E.
Fig. 7.
30
imax
(normalized at pH 7)
3.0
A
2.0
1.0
0.0
B
KM
(mM)
0.6
0.4
0.2
0.0
5
6
7
8
9
10
pH
Fig. 8.
31
Fig.9.
32