TY - JOUR
T1 - Complex formation of cytochrome P450cam with putidaredoxin. Evidence for protein-specific interactions involving the proximal thiolate ligand
AU - Unno, Masashi
AU - Christian, James F.
AU - Sjodin, Theodore
AU - Benson, David E.
AU - Macdonald, Iain D.G.
AU - Sligar, Stephen G.
AU - Champion, Paul M.
PY - 2002/1/25
Y1 - 2002/1/25
N2 - We have performed resonance Raman studies on ferrous NO- and CO-adducts of cytochrome P450cam and investigated the effects of diprotein complex formation with reduced putidaredoxin. We have found that the Fe-NO stretching mode of NO-P450cam can be resolved into two peaks at 551 and 561 cm-1, and the binding of putidaredoxin increases the intensity of the high frequency component. Because the Fe-NO mode has been shown to be more sensitive to the nature of the heme proximal ligand than to the distal pocket environment, such a perturbation upon putidaredoxin binding is suggestive of changes in conformation or electronic structure that affect the proximal iron-cysteine bond. In accordance with this idea, the isotope shifts for the Fe-XO stretching and Fe-X-O bending modes (X = N or C) are insensitive to the presence or absence of putidaredoxin, indicating that the geometry of the Fe-X-O unit is not significantly altered by the complex formation. On the other hand, complex formation does induce a perturbation of the low frequency heme vibrational modes, suggesting that alterations of the heme electronic structure and/or geometry take place when putidaredoxin binds. We also find that cytochrome b5 minimally affects the heme active site of the enzyme, although both putidaredoxin and cytochrome b5 bind to the same or similar site on P450cam. These observations suggest that there is a key specific interaction between P450cam and putidaredoxin, and that this interaction increases the population of a protein conformation that exhibits structural and/or electronic distortions of the heme group associated with the proximal side of the heme pocket and the S → Fe electron donation. These electronic and structural changes are potentially correlated with H-bonding to the proximal cysteine.
AB - We have performed resonance Raman studies on ferrous NO- and CO-adducts of cytochrome P450cam and investigated the effects of diprotein complex formation with reduced putidaredoxin. We have found that the Fe-NO stretching mode of NO-P450cam can be resolved into two peaks at 551 and 561 cm-1, and the binding of putidaredoxin increases the intensity of the high frequency component. Because the Fe-NO mode has been shown to be more sensitive to the nature of the heme proximal ligand than to the distal pocket environment, such a perturbation upon putidaredoxin binding is suggestive of changes in conformation or electronic structure that affect the proximal iron-cysteine bond. In accordance with this idea, the isotope shifts for the Fe-XO stretching and Fe-X-O bending modes (X = N or C) are insensitive to the presence or absence of putidaredoxin, indicating that the geometry of the Fe-X-O unit is not significantly altered by the complex formation. On the other hand, complex formation does induce a perturbation of the low frequency heme vibrational modes, suggesting that alterations of the heme electronic structure and/or geometry take place when putidaredoxin binds. We also find that cytochrome b5 minimally affects the heme active site of the enzyme, although both putidaredoxin and cytochrome b5 bind to the same or similar site on P450cam. These observations suggest that there is a key specific interaction between P450cam and putidaredoxin, and that this interaction increases the population of a protein conformation that exhibits structural and/or electronic distortions of the heme group associated with the proximal side of the heme pocket and the S → Fe electron donation. These electronic and structural changes are potentially correlated with H-bonding to the proximal cysteine.
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U2 - 10.1074/jbc.M108917200
DO - 10.1074/jbc.M108917200
M3 - Article
C2 - 11706033
AN - SCOPUS:0037169558
SN - 0021-9258
VL - 277
SP - 2547
EP - 2553
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
IS - 4
ER -