A conserved tryptophan at the ferredoxin-binding site of ferredoxin:nitrite oxidoreductase.
A conserved tryptophan at the ferredoxin-binding site of ferredoxin:nitrite oxidoreductase.
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DOI：
10.1006/abbi.1998.0630
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发表时间：
1998-06
影响因子：
3.9
通讯作者：
M. Hirasawa;M. Dose;S. Kleis-SanFrancisco;J. K. Hurley;G. Tollin;D. Knaff
中科院分区：
文献类型：
--
作者：
M. Hirasawa;M. Dose;S. Kleis-SanFrancisco;J. K. Hurley;G. Tollin;D. Knaff
用N-溴代琥珀酰亚胺（NBS）处理菠菜叶铁氧还蛋白依赖性亚硝酸盐还原酶，在每摩尔亚硝酸盐还原酶修饰的色氨酸略低于1摩尔的条件下，抑制酶的催化活性约1.5倍。80%，对底物结合或其他酶性质无任何影响。亚硝酸盐还原酶和铁氧还蛋白之间的复合物形成完全保护酶免受这种抑制。瞬态动力学测量表明，二阶速率常数为减少NBS-modified亚硝酸盐还原酶的还原铁氧还蛋白是约4倍大于所观察到的原生的，未修饰的酶。此外，通过5-脱氮核黄素自由基还原NBS修饰的亚硝酸盐还原酶显示出与用天然酶观察到的动力学模式不同的动力学模式，表明色氨酸修饰增加自由基对酶的低电位[4Fe-4S]簇的访问，降低对酶的siroheme基团的访问，或两者。被修饰的色氨酸已被鉴定为绝对保守的W 92。已经鉴定了一种也被NBS修饰的甲硫氨酸M73。因此，菠菜亚硝酸盐还原酶上的铁氧还蛋白结合位点似乎包括W 92和可能的M73，除了先前确定的R375，R556和K436。
Treatment of spinach leaf ferredoxin-dependent nitrite reductase with N-bromosuccinimide (NBS), under conditions where slightly less than 1 mol of tryptophan is modified per mole of nitrite reductase, inhibits the catalytic activity of the enzyme by ca. 80% without any effect on substrate binding or other enzyme properties. Complex formation between nitrite reductase and ferredoxin completely protects the enzyme against this inhibition. Transient kinetic measurements show that the second-order rate constant for reduction of NBS-modified nitrite reductase by reduced ferredoxin is approximately four-fold larger than that observed for the native, unmodified enzyme. Also, reduction of NBS-modified nitrite reductase by the 5-deazariboflavin radical shows a different kinetic pattern than that observed with the native enzyme, suggesting that tryptophan modification increases access of the radical to the low-potential [4Fe-4S] cluster of the enzyme, decreases the accessibility to the siroheme group of the enzyme, or both. The tryptophan that is modified has been identified as the absolutely conserved W92. A methionine, M73, that is also modified by NBS, has been identified. The ferredoxin-binding site on spinach nitrite reductase thus appears to include W92 and perhaps M73, in addition to the previously identified R375, R556, and K436.