Tunnel mutagenesis and Ni-dependent reduction and methylation of the alpha subunit of acetyl coenzyme A synthase/carbon monoxide dehydrogenase.
Tunnel mutagenesis and Ni-dependent reduction and methylation of the alpha subunit of acetyl coenzyme A synthase/carbon monoxide dehydrogenase.
复制标题
乙酰辅酶 A 合酶/一氧化碳脱氢酶 α 亚基的隧道诱变和 Ni 依赖性还原和甲基化。
DOI：
10.1007/s00775-008-0363-x
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发表时间：
2008
期刊：
影响因子：
--
通讯作者：
Lindahl,PaulA
中科院分区：
文献类型：
--
作者：
Tan,Xiangshi;Lindahl,PaulA
Two isolated α subunit mutants (A110C and A222L) of the α2β2acetyl coenzyme A synthase (ACS)/carbon monoxide dehydrogenase (CODH) fromMoorella thermoaceticawere designed to block the CO-migrating tunnel in the α subunit, allowing comparison with equivalent mutants in ACS/CODH. After Ni activation, both mutants exhibited electron paramagnetic resonance spectra indicating that the A-cluster was properly assembled. ACS activities were similar to those of the wild-type recombinant Ni-activated α subunit, suggesting that CO diffuses directly to the A-cluster from solvent rather than through the tunnel as is observed for the “majority” activity of ACS/CODH. Thus, CO appears to migrate to the A-cluster through two pathways, one involving and one not involving the tunnel. The kinetics and extent of reduction of the Fe4S4cubane in the apo-α subunit and the Ni-activated α subunit upon exposure to titanium(III) citrate were examined using the stopped-flow method. The extent of reduction was independent of Ni, whereas the kinetics of reduction was Ni-dependent. Apo-α subunit reduction was monophasic while Ni-activated α subunit reduction was biphasic, with the more rapid phase coincident with that of apo-α subunit reduction. Thus, binding of Ni to the A-cluster slows the reduction kinetics of the [Fe4S4]2+cubane. An upper limit of two electrons per α subunit are transferred from titanium(III) citrate to the Ni subcomponent of the A-cluster during reductive activation. These electrons are accepted quickly relative to the reduction of the [Fe4S4]2+cubane. This reduction is probably a prerequisite for methyl group transfer. CO appears to bind to reduced nonfunctional subunits, thereby inhibiting reduction (or promoting reoxidation) of the cubane subcomponent of the A-cluster.
