Biochemical and crystallographic studies of the Met144Ala, Asp92Asn and His254Phe mutants of the nitrite reductase from Alcaligenes xylosoxidans provide insight into the enzyme mechanism
Biochemical and crystallographic studies of the Met144Ala, Asp92Asn and His254Phe mutants of the nitrite reductase from Alcaligenes xylosoxidans provide insight into the enzyme mechanism
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DOI：
10.1006/jmbi.2001.5304
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发表时间：
2002-02-08
影响因子：
5.6
通讯作者：
Hasnain, SS
中科院分区：
文献类型：
--
作者：
Ellis, MJ;Prudêncio, M;Hasnain, SS
异化亚硝酸盐还原酶催化亚硝酸盐（NO2-）还原为一氧化氮（NO）。含铜亚硝酸盐还原酶含有I型和2型Cu位点。从氧化还原伙伴的电子转移被认为是介导的1型铜网站和使用的催化2型铜中心沿着与基板亚硝酸盐。在2型Cu位点，Asp92已被确定为底物利用的关键残基，因为它在亚硝酸盐结合位点与水分子形成氢键。我们还提出，质子通过Asp92进入催化位点，通过His254介导的水网络。这些残基在木糖氧化产碱杆菌的蓝色亚硝酸铜还原酶中的作用已经被研究（NCIMB 11015）的突变。此外，已经提出，该酶通过有序机制进行操作，其中当第二底物亚硝酸盐结合时，电子大部分转移到2型Cu位点，当其负载有亚硝酸盐时，通过降低2型位点的氧化还原电位来控制。因此，I型Cu位点的小扰动应导致对酶活性的显著影响。出于这个原因，Met144的突变，这是最弱的配体的1型铜，进行了研究。H254F、D92N和M144A的结构已分别被确定为1.85埃、1.9埃和2.2埃分辨率。D92N和H254F突变体具有可忽略的活性或没有活性，而M144A突变体具有与天然酶相似的30%的活性。结构和光谱数据表明，H254 F中活性的丧失是由于催化位点被Zn占据，而D92 N/M144 A中活性的丧失/降低是由于结构原因。D92N突变导致Asp92与Cu连接的水的氢键的丧失。因此，配体不再能够进行质子提取。尽管H254F的活性丧失是由于缺乏催化性Cu，但突变确实导致了水网络的破坏，证实了其在质子通道中的关键作用。H254F突变体的结构是第一种情况下，在2型铜网站完全占用锌被观察到，但尽管前面提到的相似性，该网站的碳酸酐酶催化位点，没有碳酸酐酶活性观察。的H254F和D92N突变体结构提供了，第一次，观察表面锌网站，可以作为一个锌汇，并防止在天然酶的催化铜网站的锌结合。(C)，2002 Elsevier Science Ltd.
Dissimilatory nitrite reductase catalyses the reduction of nitrite (NO2-) to nitric oxide (NO). Copper-containing nitrite reductases contain both type I and type 2 Cu sites. Electron transfer from redox partners is presumed to be mediated via the type 1 Cu site and used at the catalytic type 2 Cu centre along with the substrate nitrite. At the type 2 Cu site, Asp92 has been identified as a key residue in substrate utilisation, since it hydrogen bonds to the water molecule at the nitrite binding site. We have also suggested that protons enter the catalytic site via Asp92, through a water network that is mediated by His254. The role of these residues has been investigated in the blue copper nitrite reductase from Alcaligenes xylosoxidans (NCIMB 11015) by a combination of point mutation, enzymatic activity measurement and structure determination.In addition, it has been suggested that the enzyme operates via an ordered mechanism where an electron is transferred to the type 2 Cu site largely when the second substrate nitrite is bound and that this is controlled via the lowering of the redox potential of the type 2 site when it is loaded with nitrite. Thus, a small perturbation of the type I Cu site should result in a significant effect on the activity of the enzyme. For this reason a mutation of Met144, which is the weakest ligand of the type 1 Cu, is investigated. The structures of H254F, D92N and M144A have been determined to 1.85 Angstrom, 1.9 Angstrom and 2.2 Angstrom resolution, respectively. The D92N and H254F mutants have negligible or no activity, while the M144A mutant has similar to30% activity of the native enzyme. Structural and spectroscopic data show that the loss of activity in H254F is due to the catalytic site being occupied by Zn while the loss/reduction of activity in D92N/M144A are due to structural reasons. The D92N mutation results in the loss of the Asp92 hydrogen bond to the Cu-ligated water. Therefore, the ligand is no longer able to perform proton abstraction. Even though the loss of activity in H254F is due to lack of catalytic Cu, the mutation does cause the disruption of the water network, confirming its key role in proton channel. The structure of the H254F mutant is the first case where full occupancy Zn at the type 2 Cu site is observed, but despite the previously noted similarity of this site to the carbonic anhydrase catalytic site, no carbonic anhydrase activity is observed. The H254F and D92N mutant structures provide, for the first time, observation of surface Zn sites which may act as a Zn sink and prevent binding of Zn at the catalytic Cu site in the native enzyme. (C), 2002 Elsevier Science Ltd.