A Comparative Analysis of the Effector Role of Redox Partner Binding in Bacterial P450s.
A Comparative Analysis of the Effector Role of Redox Partner Binding in Bacterial P450s.
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DOI：
10.1021/acs.biochem.6b00913
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发表时间：
2016-11-29
期刊：
影响因子：
2.9
通讯作者：
Poulos TL
中科院分区：
文献类型：
--
作者：
Batabyal D;Lewis-Ballester A;Yeh SR;Poulos TL
樟脑单加氧酶，细胞色素P450 cam，表现出严格的要求，其自己的氧化还原伙伴，putidaredoxin（Pdx），一个两个铁硫铁氧还蛋白。与P450 cam最接近的同系物CYP 101 D1在结构上非常相似，使用相似的氧化还原配偶体，并且在樟脑的单氧化中表现出几乎相同的酶性质，以产生相同的单一5-外羟基樟脑产物。然而，CYP 101 D1并不严格要求其自身的铁氧还蛋白（Arx）的活性，因为Pdx可以支持CYP 101 D1催化，但Arx不能支持P450 cam催化。我们进一步研究了这两个P450之间的差异，通过确定自旋平衡，氧化还原性质，和氧络合物的稳定性的影响。我们发现Arx使自旋态平衡向高自旋方向移动，这与Pdx对P450 cam的影响相反。在两种P450中，氧化还原伴侣结合使氧-P450复合物不稳定，但这种作用在CYP 101 D1中弱得多。此外，共振拉曼数据显示，加入Pdx后在P450 cam中观察到的结构扰动在CYP 101 D1中不存在。这些数据表明，Arx在催化中不发挥与Pdx与P450 cam相同的效应器作用。这两种P450之间最相关的结构差异集中在质子耦合电子转移所需的催化重要的Asp残基上。我们假设，与P450凸轮更大的Pdx辅助运动需要释放这种Asp的催化，而在CYP 101 D1的限制较少的数量排除了需要氧化还原伴侣辅助的结构变化。
The camphor monooxygenase, cytochrome P450cam, exhibits a strict requirement for its own redox partner, putidaredoxin (Pdx), a two-iron–sulfur ferredoxin. The closest homologue to P450cam, CYP101D1, is structurally very similar, uses a similar redox partner, and exhibits nearly identical enzymatic properties in the monooxygenation of camphor to give the same single 5-exo-hydroxy camphor product. However, CYP101D1 does not strictly require its own ferredoxin (Arx) for activity because Pdx can support CYP101D1 catalysis but Arx cannot support P450cam catalysis. We have further examined the differences between these two P450s by determining the effect of spin equilibrium, redox properties, and stability of oxygen complexes. We find that Arx shifts the spin state equilibrium toward high-spin, which is the opposite of the effect of Pdx on P450cam. In both P450s, redox partner binding destabilizes the oxy–P450 complex but this effect is much weaker with CYP101D1. In addition, resonance Raman data show that structural perturbations observed in P450cam upon addition of Pdx are absent in CYP101D1. These data indicate that Arx does not play the same effector role in catalysis as Pdx does with P450cam. The most relevant structural difference between these two P450s centers on a catalytically important Asp residue required for proton-coupled electron transfer. We postulate that with P450cam larger Pdx-assisted motions are required to free this Asp for catalysis while the smaller number of restrictions in CYP101D1 precludes the need for redox partner-assisted structural changes.