Van Stappen Casey, Reijerse Edward, Chabbra Sonia, Schnegg Alexander, Lu Yi
Department of Chemistry, University of Texas at Austin, 105 E 24th St., Austin, TX, 78712, USA.
Max Planck Institute for Chemical Energy Conversion, Stiftstrasse 34-36, 45470, Mülheim an der Ruhr, Germany.
J Biol Inorg Chem. 2025 May 24. doi: 10.1007/s00775-025-02116-x.
Metalloproteins tune the electronic properties of metal active sites through a combination of primary and secondary coordination sphere effects (PCS and SCS) to efficiently perform an array of redox chemistry, including electron transfer (ET) and catalysis. A major influence of these effects is the anisotropic spatial distribution of redox-active molecular orbitals (RAMOs), which in turn dictates redox chemistry of the metalloproteins. While much progress has been made in understanding the SCS effects on RAMOs in individual native metalloproteins, it has been difficult to experimentally examine the influence of the same SCS effects on RAMOs with different spatial distributions. Taking advantage of our recent studies of SCS effect on blue copper azurin from Pseudomonas aeruginosa (Blue CuAz) and its M121H/H46E variant that closely mimic the red copper protein (Red CuAz), in which their RAMOs are dominated by either Cu-S or Cu-S interactions, respectively, we herein compare and contrast how the same SCS modifications impact the electronic and geometric structures of blue and red Cu center in the same protein scaffold. Specifically, we expand our understanding of unpaired electron distribution at the Cu-binding site of Red CuAz and its SCS N47S, F114P, and F114N mutations using H and N electron-nuclear double resonance (ENDOR) spectroscopy, and then further combine these data sets with recent studies and DFT calculations to provide insight into how these mutations differentially (or similarly) impact electronic structure in Red vs. Blue CuAz. We find that electrostatics produce similar effects in both Red and Blue CuAz, where the introduction of dipole moments in the vicinity of Cu and S produces changes in spin density distribution and of the same sign and comparable magnitude. However, disruption of H-bonding with S through the F114P mutation leads to opposing effects in Red vs. Blue CuAz, which we propose arise from differences in the conformation of Cys112 sidechain adapted in the absence the stabilizing S⋯H-N backbone interaction.
金属蛋白通过一级和二级配位球效应（PCS和SCS）的组合来调节金属活性位点的电子性质，从而有效地进行一系列氧化还原化学反应，包括电子转移（ET）和催化作用。这些效应的一个主要影响是氧化还原活性分子轨道（RAMOs）的各向异性空间分布，这反过来又决定了金属蛋白的氧化还原化学性质。虽然在理解单个天然金属蛋白中SCS对RAMOs的影响方面已经取得了很大进展，但很难通过实验研究相同的SCS效应对具有不同空间分布的RAMOs的影响。利用我们最近对铜绿假单胞菌蓝铜天青蛋白（Blue CuAz）及其紧密模拟红铜蛋白（Red CuAz）的M121H/H46E变体中SCS效应的研究，其中它们的RAMOs分别由Cu-S或Cu-S相互作用主导，我们在此比较和对比相同的SCS修饰如何影响同一蛋白质支架中蓝铜和红铜中心的电子和几何结构。具体而言，我们使用H和N电子-核双共振（ENDOR）光谱扩展了对Red CuAz及其SCS N47S、F114P和F114N突变的Cu结合位点上未成对电子分布的理解，然后将这些数据集与最近的研究和DFT计算进一步结合，以深入了解这些突变如何不同（或相似）地影响Red与Blue CuAz中的电子结构。我们发现静电作用在Red和Blue CuAz中产生相似的效应，其中在Cu和S附近引入偶极矩会导致自旋密度分布发生变化，且符号相同、大小相当。然而，通过F114P突变破坏与S的氢键会在Red与Blue CuAz中产生相反的效应，我们认为这是由于在没有稳定的S⋯H-N主链相互作用的情况下，Cys112侧链构象的差异所致。
