Mitchell D M, Fetter J R, Mills D A, Adelroth P, Pressler M A, Kim Y, Aasa R, Brzezinski P, Malmström B G, Alben J O, Båbcock G T, Ferguson-Miller S, Gennis R B
School of Chemical Sciences, University of illinois, Urbana 61801, USA.
Biochemistry. 1996 Oct 8;35(40):13089-93. doi: 10.1021/bi961416l.
Several putative proton transfer pathways have been identified in the recent crystal structures of the cytochrome oxidases from Paracoccus denitrificans [Iwata et al. (1995) Nature 376, 660-669] and bovine [Tsukihara (1996) Science 272, 1138-1144]. A series of residues along one face of the amphiphilic transmembrane helix IV lie in one of these proton transfer pathways. The possible role of these residues in proton transfer was examined by site-directed mutagenesis. The three conserved residues of helix IV that have been implicated in the putative proton transfer pathway (Ser-201, Asn-207, and Thr-211) were individually changed to alanine. The mutants were purified, analyzed for steady-state turnover rate and proton pumping efficiency, and structurally probed with resonance Raman spectroscopy and FTIR difference spectroscopy. The mutation of Ser-201 to alanine decreased the enzyme turnover rate by half, and was therefore further characterized using EPR spectroscopy and rapid kinetic methods. The results demonstrate that none of these hydrophilic residues are essential for proton pumping or oxygen reduction activities, and suggest a model of redundant or flexible proton transfer pathways. Whereas previously reported mutants at the start of this putative channel (e.g., Asp-132-Asn) dramatically influence both enzyme turnover and coupling to proton pumping, the current work shows that this is not the case for all residues observed in this channel.
在反硝化副球菌[岩田等人（1995年），《自然》376卷，660 - 669页]和牛的细胞色素氧化酶的最新晶体结构中，已鉴定出几条假定的质子转移途径。两亲性跨膜螺旋IV一侧的一系列残基位于这些质子转移途径之一中。通过定点诱变研究了这些残基在质子转移中的可能作用。将假定质子转移途径中涉及的螺旋IV的三个保守残基（Ser - 201、Asn - 207和Thr - 211）分别替换为丙氨酸。对突变体进行纯化，分析其稳态周转速率和质子泵浦效率，并用共振拉曼光谱和傅里叶变换红外差光谱进行结构探测。将Ser - 201突变为丙氨酸使酶的周转速率降低了一半，因此使用电子顺磁共振光谱和快速动力学方法对其进行了进一步表征。结果表明，这些亲水性残基对于质子泵浦或氧还原活性都不是必需的，并提出了一个冗余或灵活的质子转移途径模型。尽管先前报道的在这个假定通道起始处的突变体（例如，Asp - 132 - Asn）对酶的周转和与质子泵浦的偶联都有显著影响，但目前的研究表明，在这个通道中观察到的所有残基并非都是如此。
