HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

J. Biol. Chem., Vol. 277, Issue 23, 20146-20150, June 7, 2002

This Article Abstract Full Text (PDF) All Versions of this Article: 277/23/20146    most recent M112202200v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Field, S. J. Articles by Richardson, D. J. Search for Related Content PubMed PubMed Citation Articles by Field, S. J. Articles by Richardson, D. J. Social Bookmarking                      What's this?

Spectral Properties of Bacterial Nitric-oxide Reductase

RESOLUTION OF pH-DEPENDENT FORMS OF THE ACTIVE SITE HEME b₃^*

Sarah J. Field, Louise Prior^§, M. Dolores Roldán^¶, Myles R. Cheesman, Andrew J. Thomson^§, Stephen Spiro, Julea N. Butt^§, Nicholas J. Watmough, and David J. Richardson^**

From the Centre for Metalloprotein Spectroscopy and Biology, Schools of  Biological Sciences and ^§ Chemical Sciences, University of East Anglia, Norwich NR4 7TJ, United Kingdom

Received for publication, December 20, 2001, and in revised form, February 27, 2002

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEEDURES RESULTS AND DISCUSSION REFERENCES

Bacterial nitric-oxide reductase catalyzes the two electron reduction of nitric oxide to nitrous oxide. In the oxidized form the active site non-heme Fe_B and high spin heme b₃ are µ-oxo bridged. The heme b₃ has a ligand-to-metal charge transfer band centered at 595 nm, which is insensitive to pH over the range of 6.0-8.5. Partial reduction of nitric-oxide reductase yields a three electron-reduced state where only the heme b₃ remains oxidized. This results in a shift of the heme b₃ charge transfer band _max to longer wavelengths. At pH 6.0 the charge transfer band _max is 605 nm, whereas at pH 8.5 it is 635 nm. At pH 6.5 and 7.5 the nitric-oxide reductase ferric heme b₃ population is a mixture of both 605- and 635-nm forms. Magnetic circular dichroism spectroscopy suggests that at all pH values examined the proximal ligand to the ferric heme b₃ in the three electron-reduced form is histidine. At pH 8.5 the distal ligand is hydroxide, whereas at pH 6.0, when the enzyme is most active, it is water.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEEDURES RESULTS AND DISCUSSION REFERENCES

Bacterial nitric-oxide reductases (NOR)¹ catalyze shown in the following Reaction 1. 

This reaction serves either as a key step in the pathway of denitrification that uses N-oxyanions and N-oxides as respiratory electron acceptors or as a way of removing cytotoxic NO (1). The capacity for NO reduction is now recognized in a phylogenetically diverse range of bacteria, which includes soil denitrifying bacteria such as Paracoccus denitrificans and pathogenic bacteria such as Neiserria meningitidis (2-4). Three classes of NOR have been identified. The two-subunit (NorCB)-dependent class from P. denitrificans, Pseudomonas stutzeri, and Rhodobacter sphaeroides, which use cytochromes c or cupredoxins as an electron donor, the single subunit (NorB) quinol-oxidizing class from Ralstonia eutropha and N. meningitidis, and the Cu_A containing quinol and cytochrome c oxidizing enzyme of Bacillus azotoformans (2, 3, 5).

Primary structure analysis, in combination with spectroscopic studies, has clearly established NORs as divergent members of the family of respiratory heme-copper oxidases. Characterization of P. denitrificans NorBC has established that the catalytic NorB subunit binds a bis-histidine-coordinated heme b that is functionally equivalent to heme a in cytochrome-c oxidase. It also binds a high spin heme b (termed b₃) that is equivalent to heme a₃ in cytochrome-c oxidase and heme o₃ of Escherichia coli cytochrome-bo₃ quinol oxidase. In cytochrome-c oxidase and quinol oxidases this heme is magnetically coupled to a copper ion (Cu_B) to form a dinuclear center, which is the site of oxygen binding and reduction. By contrast, in NOR this Cu_B is replaced with a non-heme iron, Fe_B (6-8), which is probably ligated by the three conserved histidine residues that serve as ligands to Cu_B in cytochrome-c oxidase. A fourth metal center, a covalently bound low spin heme c with histidine and methionine axial ligands, is bound by the NorC subunit. This site has no structural counterpart in cytochrome-c oxidase but is functionally equivalent to Cu_A in that it serves as a site of electron input for the respiratory complex.

Recent NOR studies have addressed the nature of the catalytic site through site-directed mutagenesis (9), redox potentiometry (8), and ligand binding studies (6). The redox potentiometry revealed that the high spin heme b₃ of the dinuclear center had a surprisingly low midpoint redox potential (E_{m(pH 7.6)} = +60 mV) (8), which imposes a large thermodynamic barrier to reduction by the low spin electron transferring heme c (E_{m(pH 7.6)} = +310 mV) and heme b (E_{m(pH 7.6)} = +345 mV). This may be a means by which reduction of the heme b₃ is avoided to prevent forming a potentially dead-end ferrous heme-nitrosyl species with NO (8). It has also been noted that reduction of the Fe_B in the dinuclear center, which occurs when three electrons are introduced to the enzyme, results in a shift in the absorption maximum of the ligand to metal charge-transfer (CT) band associated with the high spin ferric heme b₃ from ~595 to 605 nm (8). Taken together with recent resonance Raman studies of NOR (10), this observation can be accounted for by a change in the ligation from a µ-oxo bridged dinuclear center in which there is no proximal ligand to the ferric heme b₃, to a form of the ferric heme b₃ with a proximal histidine ligand and an anionic distal ligand (8).

There is currently no agreement on a model for the catalytic cycle of NOR. However, it must involve the transfer of two protons and two electrons to the active site as these are required for the reduction of two NO molecules to N₂O and H₂O (Reaction 1). It is now generally agreed that these protons are moved to the active site from the periplasm (3). In membranes this overall process is non-electrogenic because the electrons are also derived from donors located in the periplasm. A possible proton-conducting pathway that involves one or more conserved glutamate residues has emerged from site-specific mutagenesis of NOR (9).

Two recent NOR studies have reported that the spectroscopic properties of the fully oxidized enzyme are not significantly affected by pH (10, 11). These observations are perhaps surprising given the requirement of proton uptake to the dinuclear center for NO reduction. This work reports an electronic absorption and magnetic circular dichroism (MCD) spectroscopic study of NorCB from P. denitrificans at a range of pH and redox states. This has led to the identification of three spectrally distinct forms of the ferric heme b₃, which may reflect µ-oxo bridged, hydroxide-bound, and water-bound species. The latter two species are only observed in the three electron-reduced enzyme and are pH-dependent. The results may help us to elucidate the catalytic cycle of NOR.

	EXPERIMENTAL PROCEEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEEDURES RESULTS AND DISCUSSION REFERENCES

The source of the NOR used in this study was P. denitrificans strain 93.11 (ctaDI, ctaDII qoxB::kan^R) (12) grown in batch culture in minimal medium under anaerobic denitrifying conditions. The two-subunit form of the enzyme was purified essentially as described by Grönberg et al. (8).

Electronic absorbance spectra were recorded either on an Aminco DW2000 spectrophotometer or a Hitachi U3000 spectrophotometer. Room temperature magnetic circular dichroism (RT-MCD) spectra were recorded on a Jasco J-500D circular dichrograph. An Oxford Instruments super-conducting solenoid with a 25-mm room temperature bore was used to generate magnetic fields of up to 6 tesla. MCD spectral intensities depend linearly on the magnetic field at room temperature and are expressed per unit magnetic field as /H (M¹ cm¹ tesla¹).

Mediated equilibrium redox titrations of NOR were done at 20 °C in 20 mM bis-Tris propane (BTP) supplemented with 0.05% (w/v) dodecyl maltoside, 0.5 mM EDTA, and 340 mM NaCl and adjusted to the required pH. The methodology was essentially as that described by Dutton (13). Dithionite was used as the reductant and potassium ferricyanide as the oxidant. The redox mediators, each at a final concentration of 10 µM, were phenazine methosulfate (PMS), phenazine ethosulfate (PES), 5-anthraquinone 2-sulfonate, 6-anthraquinone 2,6-disulfonate, and benzylviologen. A solution of saturated quinhydrone at pH 7 was used as a redox standard (E = +295 mV). All potentials quoted are with respect to the standard hydrogen electrode.

Preparation of three electron-reduced NOR under the control of a potentiostat was achieved using a three-electrode cell configuration with a closed sample compartment thermostated at 4 °C. All manipulations were performed in an anaerobic chamber (N₂ atmosphere with O₂ at less than 2 ppm). A 200-µl sample of 25 µM NOR in 20 mM BTP, 0.05% (w/v) dodecyl maltoside, 0.34 M NaCl, and 0.5 mM EDTA at the desired pH and supplemented with a mixture of redox mediators, which included ferricyanide, PES, PMS, and 2,6-dimethylbenzaquinone, was placed in a glassy carbon pot that provided the working electrode. A Ag/AgCl reference electrode and a platinum foil counter electrode contacted the solution through a Luggin tip and Vycor frit, respectively. The potential of the carbon pot was held at +150 mV with respect to the standard hydrogen electrode for 1 h, during which time the sample was stirred, and the current flowing from the cell fell to a negligible level. After this time the sample was withdrawn from the electrochemical cell and transferred to an anaerobic cell for MCD spectroscopy.

	RESULTS AND DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEEDURES RESULTS AND DISCUSSION REFERENCES

Samples of P. denitrificans NOR were equilibrated at pH 6.5, 7.0, 7.5, 8.0, and 8.5 in BTP buffer. Samples were poised at a range of potentials between +400 and 0 mV and absorbance spectra were collected in the range of 500 to 700 nm. At each pH value the oxidized (~+400 mV) spectrum showed the characteristic / band absorption features of low spin ferric hemes in the region 520-570 nm (Fig. 1). These features have previously been assigned to the histidine/methionine-ligated c-heme of the NorC subunit and the bis-histidine-ligated b heme of the NorB subunit (14). Each sample also exhibited a clearly resolved absorption shoulder at ~595 nm. The _max and intensity of this band was not significantly affected by the pH of the bulk phase. This 595-nm feature arises from a CT band associated with ferric high spin heme b₃, which has no proximal ligand but has a distal µ-oxo bridge to the Fe_B (10, 14). Complete reduction (E = ~0 mV) of NOR at each pH led to the appearance of the intense absorption peaks at 550 and 560 nm that arise from reduction of the low spin heme c (550 nm) and heme b (560 nm) (Fig. 1). The heme b₃ 595 nm CT band disappears on reduction. Again, analysis of the reduced spectrum at each pH value revealed no substantial differences in the _max and intensity of the absorption peaks.

View larger version (23K): [in this window] [in a new window]   Fig. 1.   Effects of pH and redox state on visible absorption spectra of NOR. Spectra were recorded during mediated potentiometric titrations. Panels A-D, dashed line, fully oxidized sample; dotted line, three electron-reduced sample; solid line, fully reduced sample. Panel A, recorded at pH 6.5. Panel B, recorded at pH 7. Panel C, recorded at pH 7.5. Panel D, recorded at pH 8.5. Spectra were recorded on samples of NOR at concentrations of between 10 and 30 µM, buffered in BTP supplemented with 0.34 M NaCl, 0.5 mM EDTA, and 0.05% dodecyl maltoside.

In an earlier study of NOR (8) carried out in Tris-HCl buffer at pH 7.6, it was demonstrated that lowering the potential of the NOR sample buffer from approximately +400 to +150 mV results in the reduction of the low spin hemes (heme b and heme c) and the non-heme iron (Fe_B). In the resulting three electron-reduced species the _max of the heme b₃ CT band shifts from 595 to ~605 nm, and the extinction coefficient decreases. Thus, reduction of the Fe_B in the dinuclear center resulted in a change in the coordination environment of the heme b₃. On the basis of MCD analysis and taking into account recent resonance Raman studies (10), this coordination change is likely to be a rebinding of the proximal histidine and the breaking of the µ-oxo bridge to form a His/anion species.

In the present study the three electron-reduced state of the enzyme has been investigated at a range of pH values in BTP buffer, which allows examination over a broad range of pH values and temperatures. Chemical poising of samples at +150 mV led to the reduction of the two low spin hemes and the Fe_B, whereas the high spin heme b₃ remained oxidized. Analysis of these samples revealed that the absorption intensity of the red-shifted CT band is strongly affected by pH. The CT band is least intense at pH 6.5 and most intense at pH 8.5 (Fig. 1, A and D). This suggests that the form of the heme b₃ present at pH 8.5 is different from that present at pH 6.5. Examination of (three electron-reduced)  (fully reduced) difference spectra (Fig. 2) shows that at pH 8.5 the CT band is positioned at 605 nm. At pH 7.5 the heme b₃ appears to in a mixed form, with one population exhibiting a 605-nm CT band and a second population exhibiting a 635-nm CT band. At pH 6.5 the 635-nm form dominates the spectrum. From these data, molar extinction coefficients of _605-700 or _700-635 as 3.66 mM¹ cm¹ and 1.18 mM¹ cm¹ were calculated for the 605 and 635-nm bands, respectively. These data suggest that at different pH values the ferric heme b₃ in the three electron-reduced enzyme has a different distal ligand.

View larger version (17K): [in this window] [in a new window]   Fig. 2.   Effects of pH on the CT band of NOR in the three electron-reduced enzyme. Difference spectra were taken of the (three electron-reduced)  (fully reduced) samples. Panel A, recorded at pH 6.5. Panel B, recorded at pH 7. Panel C, recorded at pH 7.5. Panel D, recorded at pH 8.5. Spectra were recorded on samples of NOR at concentrations of between 10 and 30 µM, buffered in BTP supplemented with 0.34 M NaCl, 0.5 mM EDTA, and 0.05% dodecyl maltoside.

High spin ferric hemes give two characteristic CT bands that produce derivative-shaped features in the MCD spectrum (15, 16). The higher energy band is seen in the 600-700-nm region, and its precise wavelength is sensitive to the nature of the axial ligands (Table I). RT-MCD studies have been successfully used to assign the exogenous-bound distal ligand of histidine-ligated high spin ferric heme of E. coli cytochrome bo₃ (30). Thus, this approach was exploited to determine the likely nature of the distal ligand to NOR heme b₃ samples buffered at pH 6.0 and 8.5. Samples were poised electrochemically at +150 mV and examined by electronic absorption and RT-MCD spectroscopy. Electrochemical poising allowed the preparation in a small volume of the concentrated samples required for MCD analysis. The electronic absorption spectra clearly show that at pH 6.0 the heme b₃ is in a pure 635-nm form and at pH 8.5 in a pure 605-nm form (Fig. 3A, inset). The RT-MCD spectra show peaks in the , , and Soret regions dominated by signals from low spin ferrous heme. CT bands associated with the high spin ferric heme b₃ are seen in the 600-650-nm region of the RT-MCD spectrum. Analysis of this region shows a band at 620 nm in the pH 8.5 sample, which is characteristic of high spin heme with a proximal histidine and a distal hydroxide (His/OH). In the pH 6.0 sample this band is red-shifted to 640 nm, a position characteristic of His/H₂O-ligated high spin ferric heme (Table I). This analysis is in good agreement with the UV-visible spectroscopic analysis of the three electron-reduced NOR generated by chemical reduction and confirms that the ligand change is not an artifact of reduction with sodium dithionite. Moreover, these data suggest that direct ligation of the heme b₃ is dependent on both the redox state of the dinuclear center and on pH. In the oxidized form the heme b₃ and non-heme Fe_B are bridged by a µ-oxo group. As the non-heme Fe_B is reduced, the heme b₃ becomes ligated by hydroxide at high pH levels (~8.5) and by water at low pH levels (~6). Recent evidence from a study of carbon monoxide binding to the fully reduced (four electron-reduced) NOR suggests that these coordination states are retained by ferrous heme b₃ (31).

View larger version (29K): [in this window] [in a new window]   Fig. 3.   Visible absorption and MCD spectra of electrochemically poised three electron-reduced NOR recorded at pH 6.0 and 8.5. Panel A, visible spectra. Panel B, RT-MCD spectra. Solid line, recorded at pH 8.5; dashed line, recorded at pH 6.0. Spectra were recorded on samples of 25 µM NOR, buffered in 20 mM BTP supplemented with 0.34 M NaCl, 0.5 mM EDTA, and 0.05% dodecyl maltoside.

Quantitative analysis of the dependence of the 605- and 635-nm bands on the potential at pH 7 to obtain E°' for each redox species is made difficult by the low intensities and overlapping nature of these bands. In the fully oxidized enzyme (Fig. 4, ) the CT band is clearly positioned at 595 nm. As the dinuclear center starts to become reduced, the 595-nm band begins to decrease in intensity. At +250 mV (Fig. 4, ---) it has decreased to approximately half its original intensity, and a new band has appeared centered at 635 nm. This movement in the CT band represents a change from a µ-oxo bridged species to one where the heme b₃ is ligated by His/H₂O. The next phase of reduction from +250 mV to +140 mV (Fig. 4, ····) causes the 595-nm band to further decrease in intensity. However, this further decrease does not correspond to a further increase in the 635-nm band but to the appearance of another new band at 605 nm. This represents the formation of a His/OH-ligated heme b₃ from the µ-oxo bridged species. During the remainder of the reduction of the dinuclear center the 605 and 635-nm bands titrate at similar potentials and have completely disappeared by +22 mV with the full reduction of the heme b₃ (Fig. 4, ··). This three-phase reduction of the dinuclear center can be clearly seen when fitting the change in absorbance of the 595-nm band against potential (Fig. 5A). The data can be fitted to three n = 1 Nernst components. The first phase corresponds to the breaking of the µ-oxo bridge, between the Fe_B and the heme b₃ and the associated shift of the 595-nm band to 635 nm, to form the His/H₂O-ligated heme b₃. This rearrangement of ligation within the dinuclear center has an E°' of +325 mV. The second phase represents a change in the dinuclear center from the µ-oxo bridged species to one where the heme b₃ is His/OH-ligated and is associated with the shift in the 595-nm band to 605 nm. This phase has an E°' of +240 mV. The final phase corresponds to the full reduction of both species from a ferric heme b₃ to a ferrous heme b₃ where the shoulder of the 605 and 635 bands detected at 595 nm disappear. Both processes occur at isopotentials where the E°' is +50 mV (Fig 6). By contrast, at pH 8.5, where only a single species is present (His/OH) in the three electron-reduced form, a simple two-phase titer is seen (Fig. 5B). The first phase represents the reduction of the Fe_B and breaking of the µ-oxo bridge and the second phase represents the full reduction of the high spin His/OH-ligated heme b₃.

View larger version (12K): [in this window] [in a new window]   Fig. 4.   Effects of potential on the CT band of NOR at pH 7.0. Difference spectra taken at fully oxidized minus fully reduced (solid line); +250 mV minus fully reduced (dashed line); +140 mV minus fully reduced (dotted line); +90 mV minus fully reduced (dot and dash line); and +22 mV minus fully reduced (dash and double dot line). Spectra were recorded on samples of NOR buffered in 20 mM BTP supplemented with 0.34 M NaCl, 0.5 mM EDTA, and 0.05% dodecyl maltoside, pH 7.

View larger version (19K): [in this window] [in a new window]   Fig. 5.   Mediated redox titration of the dinuclear center of NOR. A, titration at pH 7.0. The data fitted to two n = 1 Nernstian components with Em values of +299 mV (85%) and +62 mV (15%) is shown by the dashed line. Data fitted to three n = 1 Nernstian components with Em values of +325 mV (53%), +240 mV (32%), and +50 mV (15%) is shown by the solid line. B, titration at pH 8.5 fitted to two n = 1 Nernstian components with Em values of +150 mV (50%) and +20 mV (50%).

View larger version (15K): [in this window] [in a new window]   Fig. 6.   Diagram showing a model for the effects of pH and redox state on ligation of the high spin heme b3 of NOR. Midpoint redox potentials correspond to those observed at pH 7.0.

Having identified three spectral forms of the ferric heme b₃, it is possible to forward tentative suggestions as to both their origin and their role in possible catalytic cycles of the enzyme. At present there is no agreement on the mechanism of NO reduction by NOR. Arguments have been put forward for the two substrate NO molecules binding to Fe_B in the so-called cis model (2). An alternative trans mechanism where one NO molecule binding to each of Fe_B and heme b₃ has also been forwarded (2). However, in both cases it is likely that at some point in the catalytic cycle a ferric heme b₃ bound by a water molecule is formed. The identification of the 635-nm spectral form of heme b₃ is the first time such a species has been identified. It can be supposed that this can be derived from the reduction of a µ-oxo bridged Fe(III)-Fe(III) dinuclear center, which would correlate to the 595-nm form of oxidized NOR. It then seems plausible that on reduction of the Fe_B of the dinuclear center with one electron a proton will also enter the catalytic site leading to a hydroxide-bound ferric heme b₃ (Fig. 6). MCD studies of this spectral form of the heme b₃ are consistent with this assertion. There are many examples of high spin hemes that exhibit CT bands at ~630 nm, and these characteristically arise from ferric hemes with His-H₂O coordination (e.g. in E. coli cytochrome-bo₃ oxidase (16)). Thus it seems most likely that the low pH 635-nm form of heme b₃ arises from a simple protonation of the putative hydroxide-bound high pH 605-nm form (Fig. 6). It is also important to note that experiments in our laboratory have shown that NOR activity in BTP buffer at pH 6.0 is 8-fold higher (~70 s¹) than at pH 8.5 (~8 s¹) (data not shown). The acidic nature of this optimal activity is in agreement with previously published data obtained in various buffers (32, 33). At this pH the His-H₂O form of the enzyme dominates. The higher midpoint redox potential of this species than that of the His-hydroxide form may allow more rapid electron transfer from physiological electron donors such as cytochrome c and pseudoazurin.

Having identified spectral signals that might correspond to distinct intermediates in the catalytic cycle of NOR, it is now necessary to try to trap these spectral forms in rapid-reaction experiments. Such experiments may in turn provide further insight into the N₂O-generating half of the catalytic cycle.

	ACKNOWLEDGEMENTS

We thank Ann Reilly, Jeremy Thornton, and David Clark for expert technical contributions and Gareth Butland for helpful discussions. We also thank the EU SENORA groups of Simon de Vries, Matti Saraste, Rob van Spanning, and Costos Varotsis for useful discussions.

	FOOTNOTES

^* This work was supported by European Union SENORA Grant B104-CT98-0507 (to M. D. R), a Biotechnology and Biological Sciences Research Council Grant 83/C13457, and an Engineering and Physical Sciences Research Council studentship (to L. P.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^¶ Present address: Dept. Bioquímica y Biologia Molecular, Campus de Rabanales, Edificio C6, 1^a planta, Universidad de Córdoba, 14071, Córdoba, Spain.

A Wellcome Trust University Award lecturer (054798/Z/98/Z).

^** To whom correspondence should be addressed. Tel.: 44-1603-593250; Fax: 44-1603-592250; E-mail: d.richardson@uea.ac.uk.

Published, JBC Papers in Press, March 18, 2002, DOI 10.1074/jbc.M112202200

	ABBREVIATIONS

The abbreviations used are: NOR, nitric-oxide reductase; CT, charge transfer; RT-MCD, room temperature magnetic circular dichroism; BTP, bis-Tris propane; PMS, phenazine methosulfate; PES, phenazine ethosulfate..

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEEDURES RESULTS AND DISCUSSION REFERENCES

CiteULike

Complore

Connotea

Del.icio.us

Digg

Reddit

Technorati

This article has been cited by other articles:

				J. P. Collman, Y. Yang, A. Dey, R. A. Decreau, S. Ghosh, T. Ohta, and E. I. Solomon A functional nitric oxide reductase model PNAS, October 14, 2008; 105(41): 15660 - 15665. [Abstract] [Full Text] [PDF]

				H. Kumita, K. Matsuura, T. Hino, S. Takahashi, H. Hori, Y. Fukumori, I. Morishima, and Y. Shiro NO Reduction by Nitric-oxide Reductase from Denitrifying Bacterium Pseudomonas aeruginosa: CHARACTERIZATION OF REACTION INTERMEDIATES THAT APPEAR IN THE SINGLE TURNOVER CYCLE J. Biol. Chem., December 31, 2004; 279(53): 55247 - 55254. [Abstract] [Full Text] [PDF]

				K. L. C. Gronberg, N. J. Watmough, A. J. Thomson, D. J. Richardson, and S. J. Field Redox-dependent Open and Closed Forms of the Active Site of the Bacterial Respiratory Nitric-oxide Reductase Revealed by Cyanide Binding Studies J. Biol. Chem., April 23, 2004; 279(17): 17120 - 17125. [Abstract] [Full Text] [PDF]

				R. S. Pitcher, M. R. Cheesman, and N. J. Watmough Molecular and Spectroscopic Analysis of the Cytochrome cbb3 Oxidase from Pseudomonas stutzeri J. Biol. Chem., August 23, 2002; 277(35): 31474 - 31483. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) All Versions of this Article: 277/23/20146    most recent M112202200v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Field, S. J. Articles by Richardson, D. J. Search for Related Content PubMed PubMed Citation Articles by Field, S. J. Articles by Richardson, D. J. Social Bookmarking                      What's this?