Apo-cytochrome b5 as an indicator of changes in heme accessability: preliminary studies with cytochrome P450 3A4.
Cytochrome P450s (P450 or CYP) are the largest family of hemeproteins yet characterized. X-ray crystallographic studies have shown that the heme of the P450 hemeproteins is buried in the interior of the protein molecule. Unexplored are answers to questions concerning the role of heme in the folding of newly synthesized apo-P450s and the factors that influence changes in heme accessibility following modification of the pattern of folding of the holo-P450s. We have carried out the present studies to measure changes in heme accessibility in P450s. This is an initial step to determining whether heme-binding confers structural and functional integrity and stability to a P450 molecule. Recently, we have shown that apo-high molecular weight cytochrome b5 (apo-HMWb5) is an efficient acceptor of heme when added to a preparation of purified recombinant CYP3A4. In the present work we have studied heme binding by apo-HMWb5 when mixed with a number of different hemeproteins (myoglobin, hemoglobin, catalase, CYP4A1, CYP101, and CYP3A4). These hemeproteins differ in the location of the heme (i.e., surface or internal) allowing one to study changes in structure as measured by the process of heme transfer from one protein to another. It was found that heme transfer to apo-HMWb5 occurs relatively rapidly from hemeproteins where the heme is located at or near the surface or when the hemeprotein is denatured. In contrast, heme transfer from P450s to apo-HMWb5 occurs only following modification of the P450 structure with chaotropic agents. An exception is CYP3A4 where a measurable amount of heme is transferred to apo-HMWb5 in the absence of denaturing agents. The preliminary results described here employs apo-HMWb5 as an indicator for assessing changes in heme-availability of P450s as the protein-folding of the molecule is altered.
Gilep
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Department of Biochemistry, UT Southwestern Medical Center at Dallas, 5323 Harry Hines Boulevard, Dallas, TX 75390-9038, USA.
Guryev
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Usanov
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Estabrook
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D001059:Apoproteins / Q000737:chemistry*; D002374:Catalase / Q000737:chemistry; D042926:Cytochrome P-450 CYP4A; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry; D003573:Cytochrome b Group / Q000737:chemistry*; D045303:Cytochromes b; D006418:Heme / Q000737:chemistry*; D006454:Hemoglobins / Q000737:chemistry; D006899:Mixed Function Oxygenases / Q000737:chemistry; D008970:Molecular Weight; D009211:Myoglobin / Q000737:chemistry; D011489:Protein Denaturation; D017510:Protein Folding; D013053:Spectrophotometry
D001059:Apoproteins; D003573:Cytochrome b Group; D006454:Hemoglobins; D009211:Myoglobin; D006418:Heme; D045303:Cytochromes b; D003577:Cytochrome P-450 Enzyme System; D006899:Mixed Function Oxygenases; D002374:Catalase; D042926:Cytochrome P-450 CYP4A