Sandbox 177: Difference between revisions

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NADPH-cytochrome P450 oxidoreductase (CYPOR) is a ~78kDa, multidomain flavoprotein.<ref name="5TSON">PMID:19171935</ref>  Containing three co-factors, FMN, FAD and NADPH, CYPOR is the archetype for the mammalian diflavin-containing enzyme family.<ref name="5TSON"/> Research indicates that the protein, and other FAD/FMN binding proteins, are likely the product of the fusion of two ancestral genes.<ref name="6TSON">PMID:8078947</ref>  This would account for the two distinct binding domains, FMN and FAD/NADPH, which each provide different functional capabilities to the overall protein.<ref name="6TSON"/>
NADPH-cytochrome P450 oxidoreductase (CYPOR) is a ~78kDa, multidomain flavoprotein.<ref name="5TSON">PMID:19171935</ref>  Containing three co-factors, FMN, FAD and NADPH, CYPOR is the archetype for the mammalian diflavin-containing enzyme family.<ref name="5TSON"/> Research indicates that the protein, and other FAD/FMN binding proteins, are likely the product of the fusion of two ancestral genes.<ref name="6TSON">PMID:8078947</ref>  This would account for the two distinct binding domains, FMN and FAD/NADPH, which each provide different functional capabilities to the overall protein.<ref name="6TSON"/>


Horecker first identified this protein in 1950 as NADPH-specific cytochrome c reductase, based on his assumption that it was the redox partner for cytochrome c, found in the mitochondria.<ref name="1TSON">Horecker BL. Triphosphopyridine nucleotide-cytochrome ''c'' reductase in liver. J Biol Chem 1950 April 1;183(2):593-605</ref>  However, studies in the 1960s and later showed that its main function is actually as the redox partner for cytochrome P450 in microsomal electron transport chains, thus the name change (4).<ref name="5TSON"/>
Horecker first identified this protein in 1950 as NADPH-specific cytochrome c reductase, based on his assumption that it was the redox partner for cytochrome c, found in the mitochondria.<ref name="1TSON">Horecker BL. Triphosphopyridine nucleotide-cytochrome ''c'' reductase in liver. J Biol Chem 1950 Apr 1;183(2):593-605</ref>  However, studies in the 1960s and later showed that its main function is actually as the redox partner for cytochrome P450 in microsomal electron transport chains, thus the name change.<ref name="5TSON"/><ref name="4TSON">Phillips AH, Langdon RG. Hepatic triphosphopyridine nucleotide-cytochrome c reductase: Isolation, characterization, and kinetic studies. J Biol Chem 1962 Aug 1;237:2652-60</ref>


Regulation of this protein, which is found all tissues to some extent, is largely at the transcriptional level.<ref name="3cTSON">PMID:11306680</ref>  The thyroid hormone T3 in most cases, while adrenocorticotrophic hormone acts as a regulator in a few specific cases.<ref name="3aTSON">PMID:2495435</ref><ref name="3bTSON">PMID:1737785</ref>
Regulation of this protein, which is found all tissues to some extent, is largely at the transcriptional level.<ref name="3cTSON">PMID:11306680</ref>  The thyroid hormone T3 in most cases, while adrenocorticotrophic hormone acts as a regulator in a few specific cases.<ref name="3aTSON">PMID:2495435</ref><ref name="3bTSON">PMID:1737785</ref>
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<applet load='3es9' size='200' color='black' frame='true' align='left' scene='Sandbox_177/Cyporplain/2' caption='Highlighting the chains and associated ligands of CYPOR'/>
<applet load='3es9' size='200' color='black' frame='true' align='left' scene='Sandbox_177/Cyporplain/2' caption='Highlighting the chains and associated ligands of CYPOR'/>
CYPOR is a complex, multidomain protein composed of three chains (<scene name='Sandbox_177/Cyporchaina/2'>A</scene>, <scene name='Sandbox_177/Cyporchainb/1'>B</scene>, <scene name='Sandbox_177/Cyporchainc/1'>C</scene>). It also has three different types of associated ligands; one <scene name='Sandbox_177/Cyporfmn/2'>FMN</scene>, three <scene name='Sandbox_177/Cyporfad/2'>FAD</scene> and two <scene name='Sandbox_177/Cypornadph/2'>NADPH</scene>.<ref name="5TSON"/>   
CYPOR is a complex, multidomain protein composed of three chains (<scene name='Sandbox_177/Cyporchaina/2'>A</scene>, <scene name='Sandbox_177/Cyporchainb/1'>B</scene>, <scene name='Sandbox_177/Cyporchainc/1'>C</scene>). It also has three different types of associated ligands; one <scene name='Sandbox_177/Cyporfmn/2'>FMN</scene>, three <scene name='Sandbox_177/Cyporfad/2'>FAD</scene> and two <scene name='Sandbox_177/Cypornadph/2'>NADPH</scene>.<ref name="5TSON"/> The three associated binding domains for these ligands, a connecting domain and a transmembrane anchor are the key structural elements of CYPOR.  


The N-terminus consists of a single alpha-helix that functions as a transmembrane anchor (~6kDa), holding the protein in the endoplasmic reticulum.  The portion of the protein responsible for reducing cytochrome P450 is soluble and ~66kDa.<ref name="5TSON"/>  The first 170 residues of the soluble region are very similar to those of flavodoxin, which correlates to the fact that this is the area that binds FMN.  The FAD and NADPH binding domain is located in the C-terminal section, and is very similar to the FAD domain in ferredoxin-NADP<sup>+</sup> oxidoreductase both in terms of sequence and structure.<ref name="5TSON"/>
The N-terminus consists of a single alpha-helix that functions as a transmembrane anchor (~6kDa), holding the protein in the endoplasmic reticulum.  The portion of the protein responsible for reducing cytochrome P450 is soluble and ~66kDa.<ref name="5TSON"/>  The first 170 residues of the soluble region are very similar to those of flavodoxin, which correlates to the fact that this is the area that binds FMN.  The FAD and NADPH binding domains are located closer to the C-terminus, and are very similar to the FAD domain in ferredoxin-NADP<sup>+</sup> oxidoreductase, both in terms of sequence and structure.<ref name="5TSON"/>


Between the FMN and FAD/NADPH bind domains is a connecting domain, which is a highly flexible random coil.<ref name="5TSON"/>  The hinge region is composed of 12 residues from Gly232 to Arg243, and is highly conserved among most known CYPOR molecules (ie in rats and even in yeast).<ref name="5TSON"/>  This section is presumed to be responsible for the relatively increased mobility of the FMN domain, changes to conformation and the relative orientation of the binding domains.<ref name="5TSON"/>  Because of this, it plays a key role in the transfer of electrons between FMN and FAD. <ref name="5TSON"/>   
Between the FMN and FAD/NADPH bind domains is a connecting domain, which is a highly flexible random coil.<ref name="5TSON"/>  The hinge region is composed of 12 residues from Gly232 to Arg243, and is highly conserved among most known CYPOR molecules (ie in rats and even in yeast).<ref name="5TSON"/>  This section is presumed to be responsible for the relatively increased mobility of the FMN domain, changes to conformation and the relative orientation of the binding domains.<ref name="5TSON"/>  Because of this, it plays a key role in the transfer of electrons between FMN and FAD. <ref name="5TSON"/>   
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''In vivo'' CYPOR is believe to alternate between a one and a three electron reduced form.  While the 1 electron form is fairly stable, forming a neutral blue semiquinone, it is the hydroquinone, or 3 electron form, that is able to donate electrons to the desired redox partners.
''In vivo'' CYPOR is believe to alternate between a one and a three electron reduced form.  While the 1 electron form is fairly stable, forming a neutral blue semiquinone, it is the hydroquinone, or 3 electron form, that is able to donate electrons to the desired redox partners.


As part of the microsomal electron transport system, CYPOR moves electrons from NADPH -> FAD -> FMN -> cytochrome P450. Specifically a hydride anion is moved from NADPH to the FAD.  The two electrons are then individually passed to FMN, in a process that is believed to be conformationally gated, before being passed on to cytochrome P450, again one at a time.<ref name="5TSON"/>  This reduction of cytochrome P450 allows it to function in biosynthesis and biodegradation pathways of a variety of endogenous and foreign hydrophobic substrates, including drugs and steroids.<ref name="5TSON"/><ref name="2TSON">PMID:7743131</ref>
As part of the microsomal electron transport system, CYPOR moves electrons from:
 
'''<span style="background-color:#FFA07A">NADPH</span> → <span style="background-color:#F0E68C">FAD</span> → <span style="background-color:#98FB98">FMN</span> → <span style="background-color:#AFEEEE">Cytochrome P450</span>'''
 
Specifically a hydride anion is moved from NADPH to the FAD.  The two electrons are then individually passed to FMN, in a process that is believed to be conformationally gated, before being passed on to cytochrome P450, again one at a time.<ref name="5TSON"/>  This reduction of cytochrome P450 allows it to function in biosynthesis and biodegradation pathways of a variety of endogenous and foreign hydrophobic substrates, including drugs and steroids.<ref name="5TSON"/><ref name="2TSON">PMID:7743131</ref>


Cytochrome b5, cytochrome c and heme oxygenase can also receive electrons from CYPOR.<ref name="5TSON"/>  In these cases CYPOR is functioning in the heme degradation pathway, or with monooxygenase and/or 7-dehydrocholesterol reductase in sterol synthesis.<ref name="5TSON"/>
Cytochrome b5, cytochrome c and heme oxygenase can also receive electrons from CYPOR.<ref name="5TSON"/>  In these cases CYPOR is functioning in the heme degradation pathway, or with monooxygenase and/or 7-dehydrocholesterol reductase in sterol synthesis.<ref name="5TSON"/>
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