Furukawa Y, Morishima I
Department of Molecular Engineering, Graduate School of Engineering, Kyoto University, Kyoto 606-8501, Japan.
J Biol Chem. 2001 Apr 20;276(16):12983-90. doi: 10.1074/jbc.M010217200. Epub 2001 Jan 12.
We have investigated the osmotic pressure dependence of the association between ferric cytochrome P450cam and putidaredoxin (Pdx) to gain an insight into the role of water molecules in the P450cam-reduced Pdx complexation amenable to physiological electron transfer. The association constant was evaluated from the electron transfer rates from reduced Pdx to P450cam. The natural logarithm of the association constant K(a) was linearly reduced by the osmotic pressure, and osmotic stress yields uptake of 25 waters upon association. In contrast, uptake of only 13 waters is observed from the osmotic pressure dependence of the association in the nonphysiological redox partners P450cam and oxidized Pdx. Although general protein-protein associations proceed through dehydration around the complex interface, the interfacial waters could mediate hydrogen-bonding interactions. Therefore, about 10 more interfacial waters imply an additional water-mediated hydrogen-bonding network in the P450cam.reduced Pdx complex, which does not exist in the complex with oxidized Pdx. It is also possible that the water-mediated hydrogen-bonding interactions support a high P450cam affinity for reduced (K(a) = 0.83 microm(-1)) relative to oxidized (K(a) = 0.058 microm(-1)) Pdx. This study points to a novel role of solvents in assisting redox state-dependent interaction between P450cam and Pdx.
我们研究了铁细胞色素P450cam与恶臭假单胞菌铁氧还蛋白（Pdx）之间缔合作用对渗透压的依赖性，以深入了解水分子在适合生理电子转移的P450cam还原型Pdx络合过程中的作用。通过从还原型Pdx到P450cam的电子转移速率来评估缔合常数。缔合常数K(a)的自然对数随渗透压呈线性降低，渗透压应激导致缔合时摄取25个水分子。相比之下，从非生理性氧化还原伙伴P450cam和氧化型Pdx之间缔合作用的渗透压依赖性观察到仅摄取13个水分子。尽管一般的蛋白质-蛋白质缔合是通过复合物界面周围的脱水进行的，但界面水分子可能介导氢键相互作用。因此，多出的约10个界面水分子意味着在P450cam还原型Pdx复合物中存在额外的水介导氢键网络，而在与氧化型Pdx形成的复合物中不存在。水介导的氢键相互作用也有可能支持P450cam对还原型Pdx（K(a)=0.83 μM⁻¹）相对于氧化型Pdx（K(a)=0.058 μM⁻¹）具有更高的亲和力。这项研究指出了溶剂在协助P450cam与Pdx之间依赖氧化还原状态的相互作用中的新作用。
