How the structure of the large subunit controls function in an oxygen-tolerant [NiFe]-hydrogenase.
How the structure of the large subunit controls function in an oxygen-tolerant [NiFe]-hydrogenase.
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DOI：
10.1042/bj20131520
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发表时间：
2014-03-15
期刊：
影响因子：
--
通讯作者：
Sargent F
中科院分区：
文献类型：
--
作者：
Bowman L;Flanagan L;Fyfe PK;Parkin A;Hunter WN;Sargent F
肠道沙门氏菌是一种条件致病菌，在需氧条件下产生[NiFe]-氢化酶。在本研究中，基因工程方法被用来促进这种酶的分离，称为Hyd-5。晶体结构测定的分辨率为3.2 μ m，并观察到氢化酶由相关的大小亚基组成。结构表明大亚基的His 229靠近小亚基的近端[4Fe-3S]簇。此外，His 229被观察到位于接近埋谷氨酸（Glu 73），这是保守的耐氧氢化酶。His 229和Glu 73的Hyd-5大亚基被认为是重要的氢氧化活性和耐氧机制。用丙氨酸取代His 229或Glu 73导致Hyd-5在空气中氧化氢的能力丧失。此外，发现H229 A变体已经失去了对活性的超电势要求，这在耐氧[NiFe]-氢化酶中总是观察到。这是可能的，His 229在稳定的超氧化形式的近端簇在氧的存在下的作用，它提出，谷氨酸73可以发挥支持作用，微调的化学His 229，使这一功能。氢化酶由两个亚基组成：一个大亚基和一个小亚基。在本研究中，发现大亚基中的氨基酸会影响小亚基中的辅因子，从而有助于赋予酶耐氧性。
Salmonella enterica is an opportunistic pathogen that produces a [NiFe]-hydrogenase under aerobic conditions. In the present study, genetic engineering approaches were used to facilitate isolation of this enzyme, termed Hyd-5. The crystal structure was determined to a resolution of 3.2 Å and the hydro-genase was observed to comprise associated large and small subunits. The structure indicated that His229 from the large subunit was close to the proximal [4Fe–3S] cluster in the small subunit. In addition, His229 was observed to lie close to a buried glutamic acid (Glu73), which is conserved in oxygen-tolerant hydrogenases. His229 and Glu73 of the Hyd-5 large subunit were found to be important in both hydrogen oxidation activity and the oxygen-tolerance mechanism. Substitution of His229 or Glu73 with alanine led to a loss in the ability of Hyd-5 to oxidize hydrogen in air. Furthermore, the H229A variant was found to have lost the overpotential requirement for activity that is always observed with oxygen-tolerant [NiFe]-hydrogenases. It is possible that His229 has a role in stabilizing the super-oxidized form of the proximal cluster in the presence of oxygen, and it is proposed that Glu73could play a supporting role in fine-tuning the chemistry of His229 to enable this function. A hydrogenase consists of two subunits: a large and a small subunit. In the present study, amino acids from the large subunit were found to influence a cofactor in the small subunit, such that they help to confer oxygen-tolerance to the enzyme.