Ryle M J, Seefeldt L C
Department of Chemistry and Biochemistry, Utah State University, Logan 84322, USA.
Biochemistry. 1996 Dec 10;35(49):15654-62. doi: 10.1021/bi961886f.
MgATP binding and hydrolysis are central to all reduction reactions catalyzed by nitrogenase. The iron (Fe) protein component of nitrogenase is a homodimeric protein with a bridging [4Fe-4S] cluster and two nucleotide binding sites, one on each subunit. This work presents evidence that the [4Fe-4S] cluster domain of the nitrogenase Fe protein functions as a hinge region between the two nucleotide binding domains, participating in the cooperative binding of two nucleotides. Alanine residues at position 98 (located near the [4Fe-4S] cluster) of the Azotobacter vinelandii Fe protein were changed by means of site-directed mutagenesis to Val (V) and Gly (G), and the resulting altered proteins were purified and characterized. While the wild-type and A98G Fe proteins were found to bind two nucleotides (MgATP or MgADP) with strong cooperativity (Hill coefficient of 2), the A98V Fe protein was found to bind one nucleotide with no apparent cooperativity. The binding of two nucleotides to the wild-type Fe protein is known to induce protein conformational changes which are reflected as changes in the properties of the [4Fe-4S] cluster, including a change in the redox potential of the [4Fe-4S] cluster of -120 mV for MgATP binding (-300 to -420 mV) and of -160 mV for MgADP binding (-300 to -460 mV). The binding of one nucleotide to the A98V Fe protein was found to result in only half the lowering of the redox potential, with MgATP binding resulting in a -80 mV change (-280 to -360 mV) and MgADP binding resulting in a -50 mV change (-280 to -330 mV). Results from 1H NMR, EPR, and CD spectra, along with Fe chelation rates, were all consistent with the binding of a single nucleotide to the A98V Fe protein inducing a partial conformational change. Finally, the A98V Fe protein with one nucleotide bound, still bound to the molybdenum-iron protein but did not support MgATP hydrolysis, electron transfer, or substrate reduction. A model is discussed in which the [4Fe-4S] cluster domain can be viewed as a hinge region between the two nucleotide binding domains which facilitates conformational rearrangements required for the cooperative binding of a second nucleotide.
MgATP的结合与水解是固氮酶催化的所有还原反应的核心。固氮酶的铁（Fe）蛋白组分是一种同二聚体蛋白，带有一个桥连的[4Fe-4S]簇和两个核苷酸结合位点，每个亚基上各有一个。这项工作提供了证据，表明固氮酶Fe蛋白的[4Fe-4S]簇结构域作为两个核苷酸结合结构域之间的铰链区，参与两个核苷酸的协同结合。通过定点诱变将棕色固氮菌Fe蛋白第98位（位于[4Fe-4S]簇附近）的丙氨酸残基替换为缬氨酸（V）和甘氨酸（G），然后对产生的变异蛋白进行纯化和表征。虽然发现野生型和A98G Fe蛋白以强协同性（希尔系数为2）结合两个核苷酸（MgATP或MgADP），但发现A98V Fe蛋白结合一个核苷酸时没有明显的协同性。已知两个核苷酸与野生型Fe蛋白的结合会诱导蛋白质构象变化，这表现为[4Fe-4S]簇性质的变化，包括MgATP结合时[4Fe-4S]簇的氧化还原电位变化-120 mV（从-300到-420 mV）以及MgADP结合时变化-160 mV（从-300到-460 mV）。发现一个核苷酸与A98V Fe蛋白的结合仅导致氧化还原电位降低一半，MgATP结合导致-80 mV的变化（从-280到-360 mV），MgADP结合导致-50 mV的变化（从-280到-330 mV）。1H NMR、EPR和CD光谱的结果以及铁螯合速率均与一个核苷酸与A98V Fe蛋白的结合诱导部分构象变化一致。最后，结合了一个核苷酸的A98V Fe蛋白仍与钼铁蛋白结合，但不支持MgATP水解、电子转移或底物还原。文中讨论了一个模型，其中[4Fe-4S]簇结构域可被视为两个核苷酸结合结构域之间的铰链区，它促进了第二个核苷酸协同结合所需的构象重排。
