Bandyopadhyay D, Walda K N, Grogan T M, Magde D, Traylor T G, Sharma V S
Department of Chemistry, Indian Institute of Technology, New Delhi, India.
Biochemistry. 1996 Feb 6;35(5):1500-5. doi: 10.1021/bi9518149.
The kinetics of tert-butyl isocyanide binding to the heme protein horseradish peroxidase (HRP) at 22 degrees C was examined on all time scales, from minutes to picoseconds, in aqueous borate buffer at pH 9.08. Unlike myoglobin (Mb) or hemoglobin, HRP shows two bimolecular ligand binding processes. For comparison, binding of the same ligand with Mb was measured under identical conditions. Ligand entry into the protein from the solvent in a mixing experiment is extremely slow in HRP: the bimolecular association constant is 0.04 M-1 s-1, while in Mb it is 4 x 10(3) M-1 s-1. Surprisingly, in view of that difference, picosecond and nanosecond photolyses reveal that once the ligand has reached the iron(II) site there is no difference in cage return or escape from the protein. The rate for the fastest cage return (from the contact pair) is close to 6 x 10(10) s-1 in both proteins. The rates of escape from the contact pair to form a secondary protein-caged pair are also similar: for Mb, 10 x 10(10) s-1, and for HRP, 8.5 x 10(10) s-1. The rate of rebinding from the protein-separated cage is near 4 x 10(6) s-1 in both proteins, and the rate of escape from protein to solvent is close to 3.7 x 10(6) s-1 in both. The difference between the two proteins lies in the low-millisecond time domain. After flash photolysis of HRP, there is a concentration-dependent recombination not seen in mixing experiments. This bimolecular rate constant varies slightly for different HRP preparations, being 2.6 x 10(4) or 4.0 x 10(4) M-1 s-1 in two cases, both of which are much faster than is observed in mixing experiments, namely, 0.04 M-1 s-1. In Mb, photolysis and mixing experiments consistently give the same combination rate, which is somewhat slower than the faster part of the HRP recombination. Similar measurements for the smaller ligand methyl isocyanide revealed no anomalous behavior. The interpretation proposed involves tertiary relaxation after ligand escape, which is significant in blocking the return of the large t-BuNC, but has no apparent effect on smaller ligands. Thus, HRP-t-BuNC reveals in dramatic fashion a phenomenon merely hinted at in earlier work involving the T-state binding kinetics of hemoglobin.
在22摄氏度下，于pH 9.08的硼酸盐缓冲水溶液中，对叔丁基异腈与血红素蛋白辣根过氧化物酶（HRP）结合的动力学进行了从分钟到皮秒的全时间尺度研究。与肌红蛋白（Mb）或血红蛋白不同，HRP呈现出两个双分子配体结合过程。为作比较，在相同条件下测定了相同配体与Mb的结合情况。在混合实验中，配体从溶剂进入蛋白的过程在HRP中极其缓慢：双分子缔合常数为0.04 M⁻¹ s⁻¹，而在Mb中为4×10³ M⁻¹ s⁻¹。令人惊讶的是，鉴于这种差异，皮秒和纳秒光解显示，一旦配体到达铁（II）位点，在笼回返或从蛋白中逸出方面并无差异。两种蛋白中最快的笼回返速率（来自接触对）均接近6×10¹⁰ s⁻¹。从接触对逸出形成二级蛋白笼对的速率也相似：Mb为10×10¹⁰ s⁻¹，HRP为8.5×10¹⁰ s⁻¹。两种蛋白中从蛋白分离的笼中重新结合的速率均接近4×10⁶ s⁻¹，从蛋白向溶剂逸出的速率均接近3.7×10⁶ s⁻¹。两种蛋白的差异存在于低毫秒时域。HRP经闪光光解后，出现了混合实验中未观察到的浓度依赖性重组。该双分子速率常数在不同的HRP制剂中略有不同，两种情况下分别为2.6×10⁴或4.0×10⁴ M⁻¹ s⁻¹，两者均比混合实验中观察到的速率快得多，即0.04 M⁻¹ s⁻¹。在Mb中，光解和混合实验始终给出相同的结合速率，该速率比HRP重组的较快部分稍慢。对较小配体甲基异腈的类似测量未发现异常行为。所提出的解释涉及配体逸出后的三级弛豫，这在阻止大的叔丁基异腈回返方面具有重要意义，但对较小配体没有明显影响。因此，HRP - 叔丁基异腈以显著方式揭示了一种仅在早期涉及血红蛋白T态结合动力学的工作中有所暗示的现象。
