An electrostatically driven conformational transition is involved in the mechanisms of substrate binding and cooperativity in cytochrome P450eryF

Dmitri R. Davydov, Alexandra E. Botchkareva, Santosh Kumar, You Qun He, James R. Halpert

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Abstract

The effect of ionic strength (I) on substrate-induced spin transitions and cooperativity in cytochrome P450eryF was studied. At a saturating concentration of 1-pyrenebutanol (1-PB) increasing ionic strength in the 0.06-1.2 M range promotes the formation of the high-spin state of P450, which fraction increases from 26% at 0.06 M to 75% at 1.2 M. This effect was associated with a considerable decrease in cooperativity as revealed in the 1-PB-induced spin shift. While P450eryF exhibits distinct positive cooperativity (S50 = 8.3 μM, n = 2.4) with this substrate at low ionic strength (I = 0.06 M), n decreases to 1.2 (S50 = 3.2 μM) at I = 0.66 M. Increasing ionic strength also increases the distance between the first (effector) molecule of 1-PB and the heme, as detected by the changes in the efficiency of FRET from 1-PB to the heme. The modification of Cys154 with 7-(diethylamino)-3-(4′ -maleimidyl-phenyl)-4-methylcoumarin (CPM) largely suppresses these effects of ionic strength and causes a prominent decrease 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 formed by arginine, glutamate, and aspartate residues located in helices D, E, F, and G. Our results suggest that the binding of the first substrate molecule causes an important conformational transition in the P450eryF that facilitates the substrate-induced spin shift. This transition is apparently accompanied by dissociation or rearrangement of several salt bridges in the proximity of Cys154 and modulates accessibility and hydration of the heme pocket.

Original languageEnglish (US)
Pages (from-to)6475-6485
Number of pages11
JournalBiochemistry
Volume43
Issue number21
DOIs
StatePublished - Jun 1 2004

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Ionic strength
Osmolar Concentration
Heme
Substrates
arginine glutamate
Salts
Molecules
Isoleucine
Hydration
Saccharopolyspora erythraea eryF protein
1-pyrenebutanol

All Science Journal Classification (ASJC) codes

  • Biochemistry

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An electrostatically driven conformational transition is involved in the mechanisms of substrate binding and cooperativity in cytochrome P450eryF. / Davydov, Dmitri R.; Botchkareva, Alexandra E.; Kumar, Santosh; He, You Qun; Halpert, James R.

In: Biochemistry, Vol. 43, No. 21, 01.06.2004, p. 6475-6485.

Research output: Contribution to journalArticle

Davydov, Dmitri R. ; Botchkareva, Alexandra E. ; Kumar, Santosh ; He, You Qun ; Halpert, James R. / An electrostatically driven conformational transition is involved in the mechanisms of substrate binding and cooperativity in cytochrome P450eryF. In: Biochemistry. 2004 ; Vol. 43, No. 21. pp. 6475-6485.
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abstract = "The effect of ionic strength (I) on substrate-induced spin transitions and cooperativity in cytochrome P450eryF was studied. At a saturating concentration of 1-pyrenebutanol (1-PB) increasing ionic strength in the 0.06-1.2 M range promotes the formation of the high-spin state of P450, which fraction increases from 26{\%} at 0.06 M to 75{\%} at 1.2 M. This effect was associated with a considerable decrease in cooperativity as revealed in the 1-PB-induced spin shift. While P450eryF exhibits distinct positive cooperativity (S50 = 8.3 μM, n = 2.4) with this substrate at low ionic strength (I = 0.06 M), n decreases to 1.2 (S50 = 3.2 μM) at I = 0.66 M. Increasing ionic strength also increases the distance between the first (effector) molecule of 1-PB and the heme, as detected by the changes in the efficiency of FRET from 1-PB to the heme. The modification of Cys154 with 7-(diethylamino)-3-(4′ -maleimidyl-phenyl)-4-methylcoumarin (CPM) largely suppresses these effects of ionic strength and causes a prominent decrease 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 formed by arginine, glutamate, and aspartate residues located in helices D, E, F, and G. Our results suggest that the binding of the first substrate molecule causes an important conformational transition in the P450eryF that facilitates the substrate-induced spin shift. This transition is apparently accompanied by dissociation or rearrangement of several salt bridges in the proximity of Cys154 and modulates accessibility and hydration of the heme pocket.",
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AU - Davydov, Dmitri R.

AU - Botchkareva, Alexandra E.

AU - Kumar, Santosh

AU - He, You Qun

AU - Halpert, James R.

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N2 - The effect of ionic strength (I) on substrate-induced spin transitions and cooperativity in cytochrome P450eryF was studied. At a saturating concentration of 1-pyrenebutanol (1-PB) increasing ionic strength in the 0.06-1.2 M range promotes the formation of the high-spin state of P450, which fraction increases from 26% at 0.06 M to 75% at 1.2 M. This effect was associated with a considerable decrease in cooperativity as revealed in the 1-PB-induced spin shift. While P450eryF exhibits distinct positive cooperativity (S50 = 8.3 μM, n = 2.4) with this substrate at low ionic strength (I = 0.06 M), n decreases to 1.2 (S50 = 3.2 μM) at I = 0.66 M. Increasing ionic strength also increases the distance between the first (effector) molecule of 1-PB and the heme, as detected by the changes in the efficiency of FRET from 1-PB to the heme. The modification of Cys154 with 7-(diethylamino)-3-(4′ -maleimidyl-phenyl)-4-methylcoumarin (CPM) largely suppresses these effects of ionic strength and causes a prominent decrease 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 formed by arginine, glutamate, and aspartate residues located in helices D, E, F, and G. Our results suggest that the binding of the first substrate molecule causes an important conformational transition in the P450eryF that facilitates the substrate-induced spin shift. This transition is apparently accompanied by dissociation or rearrangement of several salt bridges in the proximity of Cys154 and modulates accessibility and hydration of the heme pocket.

AB - The effect of ionic strength (I) on substrate-induced spin transitions and cooperativity in cytochrome P450eryF was studied. At a saturating concentration of 1-pyrenebutanol (1-PB) increasing ionic strength in the 0.06-1.2 M range promotes the formation of the high-spin state of P450, which fraction increases from 26% at 0.06 M to 75% at 1.2 M. This effect was associated with a considerable decrease in cooperativity as revealed in the 1-PB-induced spin shift. While P450eryF exhibits distinct positive cooperativity (S50 = 8.3 μM, n = 2.4) with this substrate at low ionic strength (I = 0.06 M), n decreases to 1.2 (S50 = 3.2 μM) at I = 0.66 M. Increasing ionic strength also increases the distance between the first (effector) molecule of 1-PB and the heme, as detected by the changes in the efficiency of FRET from 1-PB to the heme. The modification of Cys154 with 7-(diethylamino)-3-(4′ -maleimidyl-phenyl)-4-methylcoumarin (CPM) largely suppresses these effects of ionic strength and causes a prominent decrease 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 formed by arginine, glutamate, and aspartate residues located in helices D, E, F, and G. Our results suggest that the binding of the first substrate molecule causes an important conformational transition in the P450eryF that facilitates the substrate-induced spin shift. This transition is apparently accompanied by dissociation or rearrangement of several salt bridges in the proximity of Cys154 and modulates accessibility and hydration of the heme pocket.

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