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首页> 外文期刊>Dalton transactions: An international journal of inorganic chemistry >Cryoreduction EPR and ~(13)C,~(19)F ENDOR study of substrate-bound substates and solvent kinetic isotope effects in the catalytic cycle of cytochrome P450cam and its T252A mutant
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Cryoreduction EPR and ~(13)C,~(19)F ENDOR study of substrate-bound substates and solvent kinetic isotope effects in the catalytic cycle of cytochrome P450cam and its T252A mutant

机译：EPR和〜（13）C，〜（19）F ENDOR的低温还原研究在细胞色素P450cam及其T252A突变体催化循环中底物结合的亚状态和溶剂动力学同位素效应

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We recently used cryoreduction EPR/ENDOR techniques to show that a substrate can modulate the properties of both the monooxygenase active-oxygen intermediates and of the proton-delivery network which encompasses them.In the present report we use Q-band pulsed ~(19)F ENDOR (Mims 3-pulse sequence) to examine the substrate binding geometries of camphor (a),through use of the 5,5'-difluorocamphor (c),and ~(13)C ENDOR to examine the binding of 5-methylenyl camphor (b) labeled with ~(13)C at C11 (Chart 1).These probes are examined in multiple states of the catalytic cycle of P450cam and its T252A mutant.As part of this investigation we further report a new cryoreduction reaction,the reduction of a ferroheme to the EPR-visible Fe(I) state,and use it to probe the substrate binding to the EPR-silent ferroheme state.Finally we report the solvent kinetic isotope effect on the decay of the camphor complex of the hydroperoxo-ferric intermediate,the first such measurement on an individual step within the P450cam reaction cycle.Following reduction of oxyferrous-P450cam,this step is the rate-limiting step in camphor hydroxylation,and its solv-KIE of 1.8 at 190 K establishes that it involves activation of the hydroperoxo moiety by transfer of the 'second' proton of catalysis.We suggest that the finding that the heme pocket can exist in multiple substates,including multiple substrate binding locations,even in P450cam,along with the established possibility that the hydroperoxo-ferriheme intermediate can react with substrate,may explain the formation of multiple products by P450s.
机译：我们最近使用低温还原EPR / ENDOR技术显示底物可以调节单加氧酶活性氧中间体和包含它们的质子传递网络的性质。在本报告中，我们使用Q波段脉冲〜（19）。通过使用5,5'-二氟樟脑（c）和〜（13）C ENDOR来检查樟脑的底物结合几何形状（Mims 3-pulse序列），以检查樟脑的底物结合几何形状（a），并通过〜（13）C ENDOR来检查5-亚甲基的结合樟脑（b）在C11处用〜（13）C标记（图1）。在P450cam及其T252A突变体催化循环的多个状态下检查了这些探针。作为这项研究的一部分，我们进一步报告了新的低温还原反应，将铁血红素还原为EPR可见的Fe（I）状态，并用它来探测与EPR沉默的铁血红素状态结合的底物。最后，我们报道了溶剂动力学同位素对氢过氧樟脑络合物的衰变的影响。铁中间体，首先在单个步骤上进行此类测量在还原P450cam的过程中，该步骤是樟脑羟基化的限速步骤，其sol-KIE在190 K时为1.8，这表明它涉及通过过氧化氢的转移来活化氢过氧部分。我们认为血红素囊可以存在于多个亚状态中，包括多个底物结合位置，甚至在P450cam中，以及氢过氧-铁血红素中间体可以与底物发生反应的可能性，这一发现也可以解释其形成。 P450的多种产品。

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