Earlier CO flow-flash experiments on the fully reduced Thermus thermophilus ba3 (Tt ba3) cytochrome oxidase revealed that O2 binding was slowed down by a factor of 10 in the presence of CO (Szundi et al., 2010, PNAS 107, 21010-21015). The goal of the current study is to explore whether the long apparent lifetime (∼50 ms) of the CuB+-CO complex generated upon photolysis of the CO-bound mixed-valence Tt ba3 (Koutsoupakis et al., 2019, Acc. Chem. Res. 52, 1380-1390) affects O2 and NO binding and the ability of CuB to act as an electron donor during O-O bond splitting. The CO recombination, NO binding, and the reaction of mixed-valence Tt ba3 with O2 were investigated by time-resolved optical absorption spectroscopy using the CO flow-flash approach and photolabile O2 and NO carriers. No electron backflow was detected after photolysis of the mixed-valence CO-bound Tt ba3. The rate of O2 and NO binding was two times slower than in the fully reduced enzyme in the presence of CO and 20 times slower than in the absence of CO. The purported long-lived CuB+-CO complex did not prevent O-O bond splitting and the resulting PM formation, which was significantly faster (5-10 times) than in the bovine heart enzyme. We propose that O2 binding to heme a3 in Tt ba3 causes CO to dissociate from CuB+ in a concerted manner through steric and/or electronic effects, thus allowing CuB+ to act as an electron donor in the mixed-valence enzyme. The significantly faster O2 binding and O-O bond cleavage in Tt ba3 compared to analogous steps in the aa3 oxidases could reflect evolutionary adaptation of the enzyme to the microaerobic conditions of the T. thermophilus HB8 species.

中文翻译：

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O2和NO与嗜热栖热菌混合价ba3细胞色素c氧化酶的反应

早期对完全还原的嗜热栖热菌 ba3 (Tt ba3) 细胞色素氧化酶进行的 CO 流闪实验表明，在 CO 存在下，O2 结合减慢了 10 倍（Szundi 等，2010，PNAS 107, 21010-21015 ）。当前研究的目标是探索在 CO 结合的混合价 Tt ba3 光解时产生的 CuB+-CO 复合物是否具有较长的表观寿命（~50 ms）（Koutsoupakis 等，2019，Acc. Chem. Res. 52, 1380-1390) 影响 O2 和 NO 结合以及 CuB 在 OO 键分裂过程中充当电子供体的能力。通过时间分辨光学吸收光谱，使用 CO 流动-闪光方法和光不稳定的 O2 和 NO 载体，研究了 CO 重组、NO 结合以及混合价 Tt ba3 与 O2 的反应。在混合价 CO 结合的 Tt ba3 光解后没有检测到电子回流。O2 和 NO 结合的速度比 CO 存在下完全还原的酶慢两倍，比不存在 CO 时慢 20 倍。据称长寿命的 CuB+-CO 复合物并不能阻止 OO 键分裂，并且结果 PM 的形成，明显快于牛心酶（5-10 倍）。我们提出 O2 与 Tt ba3 中的血红素 a3 结合导致 CO 通过空间和/或电子效应以协同方式从 CuB+ 解离，从而使 CuB+ 在混合价酶中充当电子供体。与 aa3 氧化酶中的类似步骤相比，Tt ba3 中明显更快的 O2 结合和 OO 键裂解可以反映酶对 T 微需氧条件的进化适应。