Nitrite reductase: Difference between revisions
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<StructureSection load='Cca.pdb' size='350' side='right' scene='Journal:JBIC:16/Cv/2' caption='Heme-containing nitrite reductase with heme and Ca+2 ions (PDB code [[3ubr]])'> | <StructureSection load='Cca.pdb' size='350' side='right' scene='Journal:JBIC:16/Cv/2' caption='Heme-containing nitrite reductase with heme and Ca+2 ions (PDB code [[3ubr]])'> | ||
__TOC__ | |||
==Function== | |||
'''Nitrite reductase''' (NIR) catalyzes the reduction of NO<sub>2</sub> to NO. There are 2 classes of NIR: (1) A '''heme-containing cytochrome Cd type NIR'''. This enzyme contains 4 heme groups. Its d-type heme group binds NO<sub>2</sub>. (2) A '''copper-containing NIR''' which produces NO<sub>2</sub>. Under anaerobic conditions bacteria rely on the reduction of nitrogen oxide species to obtain energy. NIR is part of the nitrogen cycle used for this purpose. | '''Nitrite reductase''' (NIR) catalyzes the reduction of NO<sub>2</sub> to NO. There are 2 classes of NIR: (1) A '''heme-containing cytochrome Cd type NIR'''. This enzyme contains 4 heme groups. Its d-type heme group binds NO<sub>2</sub>. (2) A '''copper-containing NIR''' which produces NO<sub>2</sub>. Under anaerobic conditions bacteria rely on the reduction of nitrogen oxide species to obtain energy. NIR is part of the nitrogen cycle used for this purpose. | ||
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For additional details see [[CcNiR]]. | For additional details see [[CcNiR]]. | ||
'''Siroheme-containing NR''' contains siroheme which is a heme-like group used for reduction of sulfur and nitrogen. | *'''Siroheme-containing NR''' contains siroheme which is a heme-like group used for reduction of sulfur and nitrogen. | ||
'''Laue Crystal Structure of ''Shewanella oneidensis'' Cytochrome c Nitrite Reductase from a High-yield Expression System''' <ref name="Youngblut">doi 10.1007/s00775-012-0885-0</ref> | '''Laue Crystal Structure of ''Shewanella oneidensis'' Cytochrome c Nitrite Reductase from a High-yield Expression System''' <ref name="Youngblut">doi 10.1007/s00775-012-0885-0</ref> | ||
The ccNiR described here is produced by the ''Shewanella oneidensis'' bacterium, which is remarkable in its own right due to the large number of electron acceptors that it can utilize. ''Shewanella'' is a facultative anaerobe, which means that it will use oxygen if available, but in the absence of oxygen can get rid of its electrons by dumping them on a wide range of alternate acceptors, of which nitrite is only one example. To handle the electron flow ''Shewanella'' uses a large number of promiscuous <scene name='Journal:JBIC:16/Cv/8'>c-heme</scene> containing electron transfer proteins. Indeed, ''Shewanella'' is exceptionally adept at producing c-heme proteins under fast-growth conditions, which many bacteria commonly used for large-scale laboratory gene expression, such as ''E. coli'', are incapable of unless they are first extensively reprogrammed genetically. Since ''Shewanella'' can be easily grown in the lab, and can naturally and easily produce c-hemes, it is an ideal host for generating large quantities of c-heme proteins such as ccNiR. | The ccNiR described here is produced by the ''Shewanella oneidensis'' bacterium, which is remarkable in its own right due to the large number of electron acceptors that it can utilize. ''Shewanella'' is a facultative anaerobe, which means that it will use oxygen if available, but in the absence of oxygen can get rid of its electrons by dumping them on a wide range of alternate acceptors, of which nitrite is only one example. To handle the electron flow ''Shewanella'' uses a large number of promiscuous <scene name='Journal:JBIC:16/Cv/8'>c-heme</scene> containing electron transfer proteins. Indeed, ''Shewanella'' is exceptionally adept at producing c-heme proteins under fast-growth conditions, which many bacteria commonly used for large-scale laboratory gene expression, such as ''E. coli'', are incapable of unless they are first extensively reprogrammed genetically. Since ''Shewanella'' can be easily grown in the lab, and can naturally and easily produce c-hemes, it is an ideal host for generating large quantities of c-heme proteins such as ccNiR. | ||
The 2012 paper by Youngblut et al. <ref name="Youngblut">none yet</ref> describes a genetically modified ''Shewanella'' strain that can produce 20 – 40 times more ccNiR per liter of culture than the wild type bacterium. The ccNir so produced can be purified easily and in large amounts. This result is important because c-heme proteins have historically proved difficult to over-express in traditional vectors such as ''E. coli''. With large quantities of ''Shewanella'' ccNIR available, Youngblut et al <ref name="Youngblut">none yet</ref> were able to obtain the crystal structure ([[3ubr]]) and do a variety of experiments. The ccNIR consists of <scene name='Journal:JBIC:16/Cv/4'>two equal subunits</scene> (<font color='darkmagenta'><b>colored in darkmagenta</b></font> and <span style="color:lime;background-color:black;font-weight:bold;">in green</span>) with <scene name='Journal:JBIC:16/Cv/5'>five c-hemes each</scene>. In the oxidized ccNIR all central heme irons are Fe3+. They can be subsequently reduced to Fe2+ either by reducing agents or electrochemically. An important conclusion of the paper is that electrons added to ccNiR are likely <scene name='Journal:JBIC:16/Cv/6'>delocalized over several hemes</scene>, rather than localized on individual hemes. | The 2012 paper by Youngblut et al. <ref name="Youngblut">none yet</ref> describes a genetically modified ''Shewanella'' strain that can produce 20 – 40 times more ccNiR per liter of culture than the wild type bacterium. The ccNir so produced can be purified easily and in large amounts. This result is important because c-heme proteins have historically proved difficult to over-express in traditional vectors such as ''E. coli''. With large quantities of ''Shewanella'' ccNIR available, Youngblut et al <ref name="Youngblut">none yet</ref> were able to obtain the crystal structure ([[3ubr]]) and do a variety of experiments. The ccNIR consists of <scene name='Journal:JBIC:16/Cv/4'>two equal subunits</scene> (<font color='darkmagenta'><b>colored in darkmagenta</b></font> and <span style="color:lime;background-color:black;font-weight:bold;">in green</span>) with <scene name='Journal:JBIC:16/Cv/5'>five c-hemes each</scene>. In the oxidized ccNIR all central heme irons are Fe3+. They can be subsequently reduced to Fe2+ either by reducing agents or electrochemically. An important conclusion of the paper is that electrons added to ccNiR are likely <scene name='Journal:JBIC:16/Cv/6'>delocalized over several hemes</scene>, rather than localized on individual hemes. | ||
The <span style="color:yellow;background-color:black;font-weight:bold;">hemes 3-5 (colored in yellow)</span> and the hemes-2 (colored in sea-green) are six coordinate and used for electron transport only, whereas the <font color='magenta'><b>two hemes-1 (colored in magenta)</b></font> are the active sites. Electrons are believed to enter via the hemes-2, but can move between subunits. Though the physiological significance of this result is not yet known, it is possible that delocalizing the electrons keeps the active site redox-potential sufficiently high until enough electrons are accumulated that the reaction with nitrite can take place. That is, CcNIR acts like a capacitor that can store electrons until they are needed. The X-ray structure of the ccNIR reveals the architecture of this capacitor. To solve the structure a non-standard method, the Laue method, was used. This became necessary since attempts to collect a high resolution data set with monochromatic X-ray radiation were not successful. At room temperature the ccNIR crystals are susceptible to radiation damage. Freezing damaged the crystals because a suitable cryoprotectant could not be found. Single pulsed Laue crystallography with 100 ps highly intense polychromatic X-ray pulses provided a solution. A dataset was collected in a few minutes. The crystals were cooled slightly to 0 °C but not frozen. Crystal settings spanned a range of 180 °C and the crystals were orthorhombic. Therefore, a Laue dataset with very high multiplicity and good quality in terms of resolution and R<sub>merge</sub> could be collected. The structure of this ccNIR was then solved by molecular replacement using the ''E. coli'' ccNIR as a template. <scene name='Journal:JBIC:16/Cv/10'>An overlay</scene> of the ''S. oneidensis'' hemes within one monomer with the corresponding ''E. coli'' hemes reveals significant similarity. ''S. oneidensis'' <span style="color:yellow;background-color:black;font-weight:bold;">hemes 3-5</span>, | The <span style="color:yellow;background-color:black;font-weight:bold;">hemes 3-5 (colored in yellow)</span> and the hemes-2 (colored in sea-green) are six coordinate and used for electron transport only, whereas the <font color='magenta'><b>two hemes-1 (colored in magenta)</b></font> are the active sites. Electrons are believed to enter via the hemes-2, but can move between subunits. Though the physiological significance of this result is not yet known, it is possible that delocalizing the electrons keeps the active site redox-potential sufficiently high until enough electrons are accumulated that the reaction with nitrite can take place. That is, CcNIR acts like a capacitor that can store electrons until they are needed. The X-ray structure of the ccNIR reveals the architecture of this capacitor. To solve the structure a non-standard method, the Laue method, was used. This became necessary since attempts to collect a high resolution data set with monochromatic X-ray radiation were not successful. At room temperature the ccNIR crystals are susceptible to radiation damage. Freezing damaged the crystals because a suitable cryoprotectant could not be found. Single pulsed Laue crystallography with 100 ps highly intense polychromatic X-ray pulses provided a solution. A dataset was collected in a few minutes. The crystals were cooled slightly to 0 °C but not frozen. Crystal settings spanned a range of 180 °C and the crystals were orthorhombic. Therefore, a Laue dataset with very high multiplicity and good quality in terms of resolution and R<sub>merge</sub> could be collected. The structure of this ccNIR was then solved by molecular replacement using the ''E. coli'' ccNIR as a template. <scene name='Journal:JBIC:16/Cv/10'>An overlay</scene> of the ''S. oneidensis'' hemes within one monomer with the corresponding ''E. coli'' hemes reveals significant similarity. ''S. oneidensis'' <span style="color:yellow;background-color:black;font-weight:bold;">hemes 3-5</span>, hemes-2, and <font color='magenta'><b>hemes-1</b></font> are colored in <span style="color:yellow;background-color:black;font-weight:bold;">yellow</span>, sea-green, and <font color='magenta'><b>magenta</b></font>, respectively, whereas their corresponding ''E. coli'' hemes are in similar, but darker colors. The <scene name='Journal:JBIC:16/Cv/11'>overall structure</scene> of ''S. oneidensis'' ccNiR also is similar to that of ''E. coli'' ccNiR, except in the region where the enzyme interacts with its physiological electron donor (CymA in the case of ''S. oneidensis'' ccNiR, NrfB in the case of the ''E. coli'' protein) near heme 2. Subunits of ''S. oneidensis'' ccNiR <font color='darkmagenta'><b>colored in darkmagenta</b></font> and <span style="color:lime;background-color:black;font-weight:bold;">in green</span>; subunits of ''E. coli'' ccNiR <span style="color:hotpink;background-color:black;font-weight:bold;">colored in hot pink</span> and <span style="color:deepskyblue;background-color:black;font-weight:bold;">in deep-sky-blue</span>. | ||
Protein film voltammetry (PFV) experiments performed on ''S. oneidensis'' ccNiR films in the absence of substrate produced a broad envelope of reversible signals that span approximately 450 mV. At high pH values the envelope appears as a single peak, whereas at pH values below 7 the envelope appears to be composed of two large overlapping peaks. At pH values below 6 and at 0 °C, the envelope of signal can be better resolved and more than two peaks can be observed. This resulting envelope of signal can be deconvoluted as the sum of five one-electron peaks, each corresponding to one of the five hemes in a ccNiR monomer (see image below). | Protein film voltammetry (PFV) experiments performed on ''S. oneidensis'' ccNiR films in the absence of substrate produced a broad envelope of reversible signals that span approximately 450 mV. At high pH values the envelope appears as a single peak, whereas at pH values below 7 the envelope appears to be composed of two large overlapping peaks. At pH values below 6 and at 0 °C, the envelope of signal can be better resolved and more than two peaks can be observed. This resulting envelope of signal can be deconvoluted as the sum of five one-electron peaks, each corresponding to one of the five hemes in a ccNiR monomer (see image below). | ||
[[Image:figur5.jpg|left|378px|thumb|PFV of ''S. oneidensis'' ccNiR (a) Typical signal on a graphite electrode. (b) Baselinesubtracted non-turnover voltammogram]] | [[Image:figur5.jpg|left|378px|thumb|PFV of ''S. oneidensis'' ccNiR (a) Typical signal on a graphite electrode. (b) Baselinesubtracted non-turnover voltammogram]] | ||
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The Ca<sup>2+</sup> ion within <scene name='Journal:JBIC:16/Cv/14'> the conserved site</scene> is coordinated in bidentate fashion by <scene name='Journal:JBIC:16/Cv/15'>Glu205</scene>, and in monodentate fashion by the <scene name='Journal:JBIC:16/Cv/16'>Tyr206 and Lys254</scene> backbone carbonyls, and the <scene name='Journal:JBIC:16/Cv/17'>Gln256</scene> side-chain carbonyl. In the ''S. oneidensis'' structure only <scene name='Journal:JBIC:16/Cv/18'>one water molecule</scene> is assigned to the Ca<sup>2+</sup> ion in subunit B. In subunit A the difference electron density that represents this water molecule is very close to the noise level, and it is difficult to identify even one water molecule there. The <scene name='Journal:JBIC:16/Cv/14'>carbonyl side chain of Asp242 and the hydroxyl of Tyr235</scene> come near to the open calcium coordination sites, but are not within bonding distance. Instead they interact with the water molecule that is weakly coordinated to the Ca<sup>2+</sup> ion. The ccNiR calcium ions appear to play a vital role in organizing the <scene name='Journal:JBIC:16/Cv/13'>active site</scene> (as was mentioned above <font color='magenta'><b>hemes-1</b></font> are the active sites). | The Ca<sup>2+</sup> ion within <scene name='Journal:JBIC:16/Cv/14'> the conserved site</scene> is coordinated in bidentate fashion by <scene name='Journal:JBIC:16/Cv/15'>Glu205</scene>, and in monodentate fashion by the <scene name='Journal:JBIC:16/Cv/16'>Tyr206 and Lys254</scene> backbone carbonyls, and the <scene name='Journal:JBIC:16/Cv/17'>Gln256</scene> side-chain carbonyl. In the ''S. oneidensis'' structure only <scene name='Journal:JBIC:16/Cv/18'>one water molecule</scene> is assigned to the Ca<sup>2+</sup> ion in subunit B. In subunit A the difference electron density that represents this water molecule is very close to the noise level, and it is difficult to identify even one water molecule there. The <scene name='Journal:JBIC:16/Cv/14'>carbonyl side chain of Asp242 and the hydroxyl of Tyr235</scene> come near to the open calcium coordination sites, but are not within bonding distance. Instead they interact with the water molecule that is weakly coordinated to the Ca<sup>2+</sup> ion. The ccNiR calcium ions appear to play a vital role in organizing the <scene name='Journal:JBIC:16/Cv/13'>active site</scene> (as was mentioned above <font color='magenta'><b>hemes-1</b></font> are the active sites). | ||
==3D structures of nitrite reductase== | ==3D structures of nitrite reductase== | ||
[[Nitrite reductase 3D structures]] | |||
</StructureSection> | |||
'''References''' | '''References''' | ||
<references/> | <references/> | ||
[[Category:Topic Page]] | [[Category:Topic Page]] | ||