Quinkal I, Davasse V, Gaillard J, Moulis J M
Département de Biologie Moléculaire et Structurale, CEN-Grenoble, France.
Protein Eng. 1994 May;7(5):681-7. doi: 10.1093/protein/7.5.681.
The widespread occurrence of Pro residues adjacent to Cys ligands in the sequences of [4Fe-4S] electron transfer proteins has not yet found a functional basis. The two such Pro of Clostridium pasteurianum 2[4Fe-4S] ferredoxin have been probed by site-directed mutagenesis. Any one of them, but not both simultaneously, can be substituted without impairing the proper folding of the protein. The reduction potentials of the ferredoxin variants fall in a narrow range of < 20 mV above the potential of the native protein. The biological activities with C. pasteurianum hydrogenase and pyruvate-ferredoxin oxidoreductase do not change significantly, except when Lys replaces Pro. In these cases, the data suggest that the two clusters of 2[4Fe-4S] ferredoxin may not always be equivalent in the interaction with the redox partners. Destabilization of the structure has been observed as the consequence of the Pro19 or Pro48 substitutions. Using 2-D NMR, this effect has been associated with perturbations of both the hydrogen bond network and one amino acid side chain around the [4Fe-4S] clusters. Thus, the conserved Pro found in the binding motif of [4Fe-4S] clusters in proteins strongly stabilizes the active site but does not play an essential role in the mechanism of electron transfer.
在[4Fe-4S]电子传递蛋白序列中，与半胱氨酸配体相邻普遍存在脯氨酸残基，但尚未发现其功能基础。通过定点诱变对巴氏梭菌2[4Fe-4S]铁氧化还原蛋白中的两个此类脯氨酸进行了探究。其中任何一个可以被取代，但不能同时取代两个，且不会损害蛋白质的正确折叠。铁氧化还原蛋白变体的还原电位比天然蛋白的电位高不到20 mV，处于较窄的范围内。与巴氏梭菌氢化酶和丙酮酸-铁氧化还原蛋白氧化还原酶的生物活性没有显著变化，除非赖氨酸取代脯氨酸。在这些情况下，数据表明2[4Fe-4S]铁氧化还原蛋白的两个簇在与氧化还原伙伴的相互作用中可能并不总是等效的。已观察到Pro19或Pro48取代导致结构不稳定。使用二维核磁共振，这种效应与[4Fe-4S]簇周围氢键网络和一个氨基酸侧链的扰动有关。因此，在蛋白质[4Fe-4S]簇结合基序中发现的保守脯氨酸强烈稳定了活性位点，但在电子传递机制中不发挥重要作用。
