An Electrostatically Driven Conformational Transition Is Involved in the Mechanisms of Substrate Binding and Cooperativity in Cytochrome P450eryF
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- 作者：Dmitri R. Davydov ; Alexandra E. Botchkareva ; Santosh Kumar ; You Qun He ; and James R. Halpert
- 刊名：Biochemistry
- 出版年：2004
- 出版时间：June 1, 2004
- 年：2004
- 卷：43
- 期：21
- 页码：6475 - 6485
- 全文大小：200K
- 年卷期：v.43,no.21(June 1, 2004)
- ISSN：1520-4995
文摘
The effect of ionic strength (I) on substrate-induced spin transitions and cooperativity incytochrome P450eryF was studied. At a saturating concentration of 1-pyrenebutanol (1-PB) increasingionic strength in the 0.06-1.2 M range promotes the formation of the high-spin state of P450, whichfraction increases from 26% at 0.06 M to 75% at 1.2 M. This effect was associated with a considerabledecrease in cooperativity as revealed in the 1-PB-induced spin shift. While P450eryF exhibits distinctpositive cooperativity (S50 = 8.3 
M, n = 2.4) with this substrate at low ionic strength (I = 0.06 M), ndecreases to 1.2 (S50 = 3.2 M) at I = 0.66 M. Increasing ionic strength also increases the distancebetween the first (effector) molecule of 1-PB and the heme, as detected by the changes in the efficiencyof FRET from 1-PB to the heme. The modification of Cys154 with 7-(diethylamino)-3-(4'-maleimidylphenyl)-4-methylcoumarin (CPM) largely suppresses these effects of ionic strength and causes a prominentdecrease in the cooperativity. The same effect was observed when Cys154 was substituted with isoleucine.Importantly, Cys154 is located at the C-terminal end of helix E and is surrounded by salt bridges formedby arginine, glutamate, and aspartate residues located in helices D, E, F, and G. Our results suggest thatthe binding of the first substrate molecule causes an important conformational transition in the P450eryFthat facilitates the substrate-induced spin shift. This transition is apparently accompanied by dissociationor rearrangement of several salt bridges in the proximity of Cys154 and modulates accessibility and hydrationof the heme pocket.
M, n = 2.4) with this substrate at low ionic strength (I = 0.06 M), ndecreases to 1.2 (S50 = 3.2 M) at I = 0.66 M. Increasing ionic strength also increases the distancebetween the first (effector) molecule of 1-PB and the heme, as detected by the changes in the efficiencyof FRET from 1-PB to the heme. The modification of Cys154 with 7-(diethylamino)-3-(4'-maleimidylphenyl)-4-methylcoumarin (CPM) largely suppresses these effects of ionic strength and causes a prominentdecrease in the cooperativity. The same effect was observed when Cys154 was substituted with isoleucine.Importantly, Cys154 is located at the C-terminal end of helix E and is surrounded by salt bridges formedby arginine, glutamate, and aspartate residues located in helices D, E, F, and G. Our results suggest thatthe binding of the first substrate molecule causes an important conformational transition in the P450eryFthat facilitates the substrate-induced spin shift. This transition is apparently accompanied by dissociationor rearrangement of several salt bridges in the proximity of Cys154 and modulates accessibility and hydrationof the heme pocket.