Access to the active site of periplasmic nitrate reductase:: Insights from site-directed mutagenesis and zinc inhibition Studies
Access to the active site of periplasmic nitrate reductase:: Insights from site-directed mutagenesis and zinc inhibition Studies
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DOI：
10.1021/bi700928m
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发表时间：
2007-08-28
期刊：
影响因子：
2.9
通讯作者：
Pignol, David
中科院分区：
文献类型：
--
作者：
Dementin, Sebastien;Arnoux, Pascal;Pignol, David
周质硝酸还原酶（NapAB）是DMSO还原酶超家族的成员，催化细菌脱氮过程的第一步。在这种异源二聚体中，二血红素NapB亚基与结合[4Fe-4S]簇和双（双蝶呤鸟嘌呤二核苷酸）辅因子的催化NapA亚基相关联。在这里，我们报告的动力学特性纯化突变异源二聚体从球形红细菌。通过结合定点诱变，氧化还原电位法，EPR光谱，和酶的特性，我们调查的催化作用的两个保守的残基（M153和R392）位于附近的钼活性位点。我们证明，M153和R392参与硝酸盐结合：在M153 A和R392 A突变体上测得的V-m与野生型酶上测得的相似，而硝酸盐的Km分别增加了10倍和200倍。另一种酶测定法的使用使我们发现NapAB被Zn 2+离子非竞争性地抑制（Ki ′ = 1 μ M）。我们利用这一特性进一步探测突变酶的活性位点。建议R392通过防止小还原分子直接还原Mo原子并部分保护活性位点免受锌抑制而充当过滤器。此外，我们表明，M153是一个关键的残基介导这种抑制可能通过协调锌离子通过其硫原子。该残基在DMSO还原酶超家族中不保守，而在周质硝酸还原酶家族中保守。因此，锌抑制可能是特异性的，并仅限于周质硝酸还原酶。
The periplasmic nitrate reductase (NapAB), a member of the DMSO reductase superfamily, catalyzes the first step of the denitrification process in bacteria. In this heterodimer, a di-heme NapB subunit is associated to the catalytic NapA subunit that binds a [4Fe-4S] cluster and a bis(molybdopterin guanine dinucleotide) cofactor. Here, we report the kinetic characterization of purified mutated heterodimers from Rhodobacter sphaeroides. By combining site-directed mutagenesis, redox potentiometry, EPR spectroscopy, and enzymatic characterization, we investigate the catalytic role of two conserved residues (M153 and R392) located in the vicinity of the molybdenum active site. We demonstrate that M153 and R392 are involved in nitrate binding: the V-m measured on the M153A and R392A mutants are similar to that measured on the wild-type enzyme, whereas the K-m for nitrate is increased 10-fold and 200-fold, respectively. The use of an alternative enzymatic assay led us to discover that NapAB is uncompetitively inhibited by Zn2+ ions (K-i' = 1 mu M). We used this property to further probe the active site access in the mutant enzymes. It is proposed that R392 acts as a filter by preventing a direct reduction of the Mo atom by small reducing molecules and partially protecting the active site against zinc inhibition. In addition, we show that M153 is a key residue mediating this inhibition likely by coordinating Zn2+ ions via its sulfur atom. This residue is not conserved in the DMSO reductase superfamily while it is conserved in the periplasmic nitrate reductase family. Zinc inhibition is therefore likely to be specific and restricted to periplasmic nitrate reductases.