Charge-Transfer Dynamics at the α/β Subunit Interface of a Photochemical Ribonucleotide Reductase.
Charge-Transfer Dynamics at the α/β Subunit Interface of a Photochemical Ribonucleotide Reductase.
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DOI：
10.1021/jacs.5b09259
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发表时间：
2016-02-03
影响因子：
15
通讯作者：
Nocera DG
中科院分区：
文献类型：
--
作者：
Olshansky L;Stubbe J;Nocera DG
核糖核苷酸还原酶（RNR）催化核糖核苷酸转化为脱氧核糖核苷酸，为DNA复制和修复提供单体结构单元。核苷酸还原通过多步质子偶联电子转移（PCET）的方式在跨越约35 kDa和两个亚基（α2和β2）的氧化还原活性氨基酸的途径上发生。尽管PCET在RNR中是快速的，但缓慢的构象变化掩盖了这些步骤的动力学检查。因此，我们开创了一种方法，在β2亚基（photoβ2）表面上位点特异性掺入[ReI]光氧化剂，允许相邻PCET途径残基β-Y356的光化学氧化，并对随后的反应性进行时间分辨光谱观察。制备了一系列能够进行光引发底物翻转的光β 2，其中掺入了四种不同的氟代酪氨酸（FnY）代替β-Y356。FnY在生物条件下去质子化，通过电子转移（ET）进行氧化，并提供一种方法来改变ET驱动力（ΔG°），同时使整个系列的额外扰动最小。我们已经使用这些特征来映射ΔG°和kET之间的相关性，无论有没有完全组装的photoRNR复合物。FnY 356在α/β亚基界面内的光氧化发生在Marcus反转区域内，重组能为λ 1 eV。我们还观察到增强的供体和受体（HDA）之间的电子耦合在一个完整的PCET途径的存在下。此外，我们还通过多种方法研究了质子转移（PT）的动力学，包括对溶剂同位素组成、缓冲液浓度和pH值的依赖性。我们提供了α2在β-Y356光氧化过程中促进PT的作用的证据; PT通过容易交换的位置和相对“紧密”的亚基界面发生。这些发现表明，RNR通过降低λ，提高HDA和在单个多肽亚基内和之间引导PT来控制ET。
Ribonucleotide reductase (RNR) catalyzes the conversion of ribonucleotides to deoxyribonucleotides to provide the monomeric building blocks for DNA replication and repair. Nucleotide reduction occurs by way of multi-step proton-coupled electron transfer (PCET) over a pathway of redox active amino acids spanning ~ 35 Å and two subunits (α2 and β2). Despite the fact that PCET in RNR is rapid, slow conformational changes mask kinetic examination of these steps. As such, we have pioneered methodology in which site-specific incorporation of a [ReI] photooxidant on the surface of the β2 subunit (photoβ2) allows photochemical oxidation of the adjacent PCET pathway residue β-Y356 and time-resolved spectroscopic observation of the ensuing reactivity. A series of photoβ2s capable of performing photoinitiated substrate turnover have been prepared in which four different fluorotyrosines (FnYs) are incorporated in place of β-Y356. The FnYs are deprotonated under biological conditions, undergo oxidation by electron transfer (ET) and provide a means by which to vary the ET driving force (ΔG°) with minimal additional perturbations across the series. We have used these features to map the correlation between ΔG° and kET both with and without the fully assembled photoRNR complex. The photooxidation of FnY356 within the α/β subunit interface occurs within the Marcus inverted region with a reorganization energy of λ ≈ 1 eV. We also observe enhanced electronic coupling between donor and acceptor (HDA) in the presence of an intact PCET pathway. Additionally, we have investigated the dynamics of proton transfer (PT) by a variety of methods including dependencies on solvent isotopic composition, buffer concentration, and pH. We present evidence for the role of α2 in facilitating PT during β-Y356 photooxidation; PT occurs by way of readily exchangeable positions and within a relatively “tight” subunit interface. These findings show that RNR controls ET by lowering λ, raising HDA, and directing PT both within and between individual polypeptide subunits.