Hydroxylamine Complexes of Cytochrome c′ : Influence of Heme Iron Redox State on Kinetic and Spectroscopic Properties
Hydroxylamine Complexes of Cytochrome c′ : Influence of Heme Iron Redox State on Kinetic and Spectroscopic Properties
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细胞色素 c 的羟胺配合物：血红素铁氧化还原状态对动力学和光谱性质的影响
DOI：
10.1021/acs.inorgchem.0c01925
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发表时间：
2020
影响因子：
4.6
通讯作者：
Andrew, Colin R.
中科院分区：
文献类型：
--
作者：
Brown, Brianna N.;Robinson, Kelsey J.;Durfee, Quentin C.;Kekilli, Demet;Hough, Michael A.;Andrew, Colin R.
羟胺（NH 2 OH或HA）是一种具有氧化还原活性的氮氧化物，在硝化细菌和甲烷氧化细菌氧化铵的过程中作为有毒中间体出现。在含铵环境中，HA由氨单加氧酶（硝化菌）或甲烷单加氧酶（甲烷氧化菌）产生。随后的HA氧化由血红素蛋白催化，包括细胞色素P460和多血红素羟胺氧化还原酶，前者导致N2 O（一种消耗臭氧层的温室气体）的排放。血红素-HA复合物也是细胞色素亚硝酸盐还原酶将亚硝酸盐还原为氨的中间体。尽管血红素-HA复合物在生物化学氮循环中的重要性，但其配位化学的基本方面仍然未知，包括Fe氧化还原状态对血红素-HA亲和力、动力学和光谱学的影响。利用停流紫外可见光谱和共振拉曼光谱，我们研究了木糖氧化产碱杆菌细胞色素α ′（L16 G AxCP-α）的L16 G远端口袋变体的HA复合物，L16 G AxCP-α是一种五配位型细胞色素，我们发现其以Fe（III）（Kd = 2.5 mM）和Fe（II）（Kd= 0.0345 mM）状态结合HA。Fe（II）状态的HA亲和力高出70倍主要是由于其较低的koff值（0.0994 s-1vs 11 s-1），而Fe（II）（2880 M-1 s-1）和Fe（III）（4300 M-1 s-1）氧化还原状态的koff值相对相似。L16 G AxCP-α的HA和咪唑亲和力的比较也用于预测Fe氧化还原状态对HA与其他蛋白质结合的影响。虽然L16 G AxCP-α的HA复合物通过氧化还原反应分解，但在过量还原剂存在下，Fe（II）HA复合物的寿命延长。确定的Fe（II）HA络合物的光谱参数包括NH_2OH配体的N-O伸缩振动，ν（N-O）= 906 cm-1。总的来说，动力学趋势和光谱基准从这项研究提供了一个基础，为今后的调查血红素-HA反应机制。
Hydroxylamine (NH2OH or HA) is a redox-active nitrogen oxide that occurs as a toxic intermediate in the oxidation of ammonium by nitrifying and methanotrophic bacteria. Within ammonium containing environments, HA is generated by ammonia monooxygenase (nitrifiers) or methane monooxygenase (methanotrophs). Subsequent oxidation of HA is catalyzed by heme proteins, including cytochromes P460 and multiheme hydroxylamine oxidoreductases, the former contributing to emissions of N2O, an ozone-depleting greenhouse gas. A heme–HA complex is also a proposed intermediate in the reduction of nitrite to ammonia by cytochromecnitrite reductase. Despite the importance of heme–HA complexes within the biogeochemical nitrogen cycle, fundamental aspects of their coordination chemistry remain unknown, including the effect of the Fe redox state on heme–HA affinity, kinetics, and spectroscopy. Using stopped-flow UV–vis and resonance Raman spectroscopy, we investigated HA complexes of the L16G distal pocket variant ofAlcaligenes xylosoxidanscytochromec′-α (L16G AxCP-α), a pentacoordinatec-type cytochrome that we show binds HA in its Fe(III) (Kd∼ 2.5 mM) and Fe(II) (Kd= 0.0345 mM) states. The ∼70-fold higher HA affinity of the Fe(II) state is due mostly to its lowerkoffvalue (0.0994 s–1vs 11 s–1), whereaskonvalues for Fe(II) (2880 M–1s–1) and Fe(III) (4300 M–1s–1) redox states are relatively similar. A comparison of the HA and imidazole affinities of L16G AxCP-α was also used to predict the influence of Fe redox state on HA binding to other proteins. Although HA complexes of L16G AxCP-α decompose via redox reactions, the lifetime of the Fe(II)HA complex was prolonged in the presence of excess reductant. Spectroscopic parameters determined for the Fe(II)HA complex include the N–O stretching vibration of the NH2OH ligand, ν(N–O) = 906 cm–1. Overall, the kinetic trends and spectroscopic benchmarks from this study provide a foundation for future investigations of heme–HA reaction mechanisms.