1iwi: Difference between revisions

Revision as of 14:16, 21 February 2008

Putidaredoxin-Binding Stablilizes an Active Conformer of Cytochrome P450cam in its Reduced State; Crystal Structure of Cytochrome P450cam

OverviewOverview

Ferrous-carbon monoxide bound form of cytochrome P450cam (CO-P450cam) has two infrared (IR) CO stretching bands at 1940 and 1932 cm(-1). The former band is dominant (>95% in area) for CO-P450cam free of putidaredoxin (Pdx), while the latter band is dominant (>95% in area) in the complex of CO-P450cam with reduced Pdx. The binding of Pdx to CO-P450cam thus evokes a conformational change in the heme active site. To study the mechanism involved in the conformational change, surface amino acid residues Arg79, Arg109, and Arg112 in P450cam were replaced with Lys, Gln, and Met. IR spectroscopic and kinetic analyses of the mutants revealed that an enzyme that has a larger 1932 cm(-1) band area upon Pdx-binding has a larger catalytic activity. Examination of the crystal structures of R109K and R112K suggested that the interaction between the guanidium group of Arg112 and Pdx is important for the conformational change. The mutations did not change a coupling ratio between the hydroxylation product and oxygen consumed. We interpret these findings to mean that the interaction of P450cam with Pdx through Arg112 enhances electron donation from the proximal ligand (Cys357) to the O-O bond of iron-bound O(2) and, possibly, promotes electron transfer from reduced Pdx to oxyP450cam, thereby facilitating the O-O bond splitting.

About this StructureAbout this Structure

1IWI is a Single protein structure of sequence from Pseudomonas putida with and as ligands. Active as Camphor 5-monooxygenase, with EC number 1.14.15.1 Full crystallographic information is available from OCA.

ReferenceReference

Infrared spectroscopic and mutational studies on putidaredoxin-induced conformational changes in ferrous CO-P450cam., Nagano S, Shimada H, Tarumi A, Hishiki T, Kimata-Ariga Y, Egawa T, Suematsu M, Park SY, Adachi S, Shiro Y, Ishimura Y, Biochemistry. 2003 Dec 16;42(49):14507-14. PMID:14661963

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-[[Image:1iwi.jpg|left|200px]]<br /><applet load="1iwi" size="450" color="white" frame="true" align="right" spinBox="true"
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 caption="1iwi, resolution 2.00&Aring;" />
 '''Putidaredoxin-Binding Stablilizes an Active Conformer of Cytochrome P450cam in its Reduced State; Crystal Structure of Cytochrome P450cam'''<br />
 ==Overview==
-Ferrous-carbon monoxide bound form of cytochrome P450cam (CO-P450cam) has, two infrared (IR) CO stretching bands at 1940 and 1932 cm(-1). The former, band is dominant (&gt;95% in area) for CO-P450cam free of putidaredoxin, (Pdx), while the latter band is dominant (&gt;95% in area) in the complex of, CO-P450cam with reduced Pdx. The binding of Pdx to CO-P450cam thus evokes, a conformational change in the heme active site. To study the mechanism, involved in the conformational change, surface amino acid residues Arg79, Arg109, and Arg112 in P450cam were replaced with Lys, Gln, and Met. IR, spectroscopic and kinetic analyses of the mutants revealed that an enzyme, that has a larger 1932 cm(-1) band area upon Pdx-binding has a larger, catalytic activity. Examination of the crystal structures of R109K and, R112K suggested that the interaction between the guanidium group of Arg112, and Pdx is important for the conformational change. The mutations did not, change a coupling ratio between the hydroxylation product and oxygen, consumed. We interpret these findings to mean that the interaction of, P450cam with Pdx through Arg112 enhances electron donation from the, proximal ligand (Cys357) to the O-O bond of iron-bound O(2) and, possibly, promotes electron transfer from reduced Pdx to oxyP450cam, thereby, facilitating the O-O bond splitting.
+Ferrous-carbon monoxide bound form of cytochrome P450cam (CO-P450cam) has two infrared (IR) CO stretching bands at 1940 and 1932 cm(-1). The former band is dominant (&gt;95% in area) for CO-P450cam free of putidaredoxin (Pdx), while the latter band is dominant (&gt;95% in area) in the complex of CO-P450cam with reduced Pdx. The binding of Pdx to CO-P450cam thus evokes a conformational change in the heme active site. To study the mechanism involved in the conformational change, surface amino acid residues Arg79, Arg109, and Arg112 in P450cam were replaced with Lys, Gln, and Met. IR spectroscopic and kinetic analyses of the mutants revealed that an enzyme that has a larger 1932 cm(-1) band area upon Pdx-binding has a larger catalytic activity. Examination of the crystal structures of R109K and R112K suggested that the interaction between the guanidium group of Arg112 and Pdx is important for the conformational change. The mutations did not change a coupling ratio between the hydroxylation product and oxygen consumed. We interpret these findings to mean that the interaction of P450cam with Pdx through Arg112 enhances electron donation from the proximal ligand (Cys357) to the O-O bond of iron-bound O(2) and, possibly, promotes electron transfer from reduced Pdx to oxyP450cam, thereby facilitating the O-O bond splitting.
 ==About this Structure==
-IWI is a [http://en.wikipedia.org/wiki/Single_protein Single protein] structure of sequence from [http://en.wikipedia.org/wiki/Pseudomonas_putida Pseudomonas putida] with HEM and CAM as [http://en.wikipedia.org/wiki/ligands ligands]. Active as [http://en.wikipedia.org/wiki/Camphor_5-monooxygenase Camphor 5-monooxygenase], with EC number [http://www.brenda-enzymes.info/php/result_flat.php4?ecno=1.14.15.1 1.14.15.1] Full crystallographic information is available from [http://ispc.weizmann.ac.il/oca-bin/ocashort?id=1IWI OCA].
+IWI is a [http://en.wikipedia.org/wiki/Single_protein Single protein] structure of sequence from [http://en.wikipedia.org/wiki/Pseudomonas_putida Pseudomonas putida] with <scene name='pdbligand=HEM:'>HEM</scene> and <scene name='pdbligand=CAM:'>CAM</scene> as [http://en.wikipedia.org/wiki/ligands ligands]. Active as [http://en.wikipedia.org/wiki/Camphor_5-monooxygenase Camphor 5-monooxygenase], with EC number [http://www.brenda-enzymes.info/php/result_flat.php4?ecno=1.14.15.1 1.14.15.1] Full crystallographic information is available from [http://oca.weizmann.ac.il/oca-bin/ocashort?id=1IWI OCA].
 ==Reference==
@@ Line 20: / Line 20: @@
 [[Category: Kimata-Ariga, Y.]]
 [[Category: Nagano, S.]]
-[[Category: Park, S.Y.]]
+[[Category: Park, S Y.]]
-[[Category: RSGI, RIKEN.Structural.Genomics/Proteomics.Initiative.]]
+[[Category: RSGI, RIKEN Structural Genomics/Proteomics Initiative.]]
 [[Category: Shimada, H.]]
 [[Category: Shiro, Y.]]
@@ Line 34: / Line 34: @@
 [[Category: structural genomics]]
-''Page seeded by [http://ispc.weizmann.ac.il/oca OCA ] on Tue Nov 20 17:43:41 2007''
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