Arciero D M, Golombek A, Hendrich M P, Hooper A B
Department of Genetics and Cell Biology, University of Minnesota, St. Paul, Minnesota 55108, USA.
Biochemistry. 1998 Jan 13;37(2):523-9. doi: 10.1021/bi972187l.
Hydroxylamine oxidoreductase (HAO) of Nitrosomonas europaea catalyzes the four-electron oxidation of NH2OH to NO2-. Each subunit of the trimeric enzyme contains seven c-hemes and one heme P460. In previous work [Hendrich, M. P., et al. (1994) J. Am. Chem. Soc. 116, 11961-11968], an integer-spin EPR signal at g = 7.7 was discovered from the active site of the resting enzyme. This new signal was assigned to an exchange-coupled cluster containing ferric heme P460 and a ferric c-heme. An electrochemical titration of HAO is presented here in which EPR signals and optical bands, believed to be associated with the P460 heme, are monitored. In the EPR titration, as a redox center with Em8 = -140 mV becomes reduced, the integer-spin signal disappears. Then, upon reduction of a redox center with Em8 = -190 mV, a g = 6 type signal, which has been previously assigned to a high-spin form of the ferric P460 heme of HAO, appears. However, in the -140 to -190 mV range, we have been unable to identify an additional EPR signal attributable to the P460 center. Thus, the electronic environment of oxidized P460 heme of HAO appears to pass through three states before reduction in a titration experiment, with an intermediate state that is not readily detectable by X-band EPR. The best candidate for the c-heme partner of the P460 heme is the heme at -190 mV, which would correspond to heme 6 of the crystal structure. A possible function of the exchange-coupled heme cluster is to facilitate two-electron oxidation steps of the substrate. An earlier spectropotentiometric titration of HAO [Collins, M. J., et al. (1993) J. Biol. Chem. 268, 14655-14662] identified a broad, weak optical band, centered near 740 nm, that was tentatively assigned to the oxidized P460 heme. This assignment has been strengthened by additional spectropotentiometric titrations at several values of pH and also by rapid kinetic experiments following the reduction of HAO by dithionite. The 740 nm band is not observed in fully oxidized HAO. In the spectropotentiometric titrations, its appearance cannot be correlated with reduction of a specific c-heme nor modeled to a Nernstian one-electron redox center. Instead, the range of potential in which the 740 nm band is present depends on whether the titration is carried out in an oxidative or reductive direction. One possible interpretation is that the 740 nm band is a property of the oxidized high-spin P460 heme but not of the low-spin state, and that the transition between the two spin states occurs at different potentials depending on the direction of the electrochemical titration.
欧洲亚硝化单胞菌的羟胺氧化还原酶（HAO）催化NH2OH到NO2-的四电子氧化反应。该三聚体酶的每个亚基包含7个c-血红素和1个血红素P460。在之前的工作中[亨德里克，M.P.等人（1994年）《美国化学会志》116，11961 - 11968]，从静息酶的活性位点发现了一个g = 7.7的整数自旋EPR信号。这个新信号被归因于一个包含铁血红素P460和一个铁c-血红素的交换耦合簇。本文展示了HAO的电化学滴定，其中监测了被认为与P460血红素相关的EPR信号和光谱带。在EPR滴定中，当一个Em8 = -140 mV的氧化还原中心被还原时，整数自旋信号消失。然后，当一个Em8 = -190 mV的氧化还原中心被还原时，一个g = 6型信号出现，该信号先前已被归因于HAO的铁P460血红素的高自旋形式。然而，在 -140至 -190 mV范围内，我们无法识别出另一个可归因于P460中心的EPR信号。因此，在滴定实验中，HAO的氧化态P460血红素的电子环境在还原之前似乎经历了三种状态，其中一种中间状态不易通过X波段EPR检测到。P460血红素的c-血红素伙伴的最佳候选者是处于 -190 mV的血红素，这将对应于晶体结构中的血红素6。交换耦合血红素簇的一个可能功能是促进底物的双电子氧化步骤。HAO的早期分光电位滴定[柯林斯，M.J.等人（1993年）《生物化学杂志》268，14655 - 14662]确定了一个以740 nm附近为中心的宽而弱的光谱带，该光谱带初步被归因于氧化态的P460血红素。通过在几个pH值下进行的额外分光电位滴定以及连二亚硫酸盐还原HAO后的快速动力学实验，这一归属得到了加强。在完全氧化的HAO中未观察到740 nm的光谱带。在分光电位滴定中，其出现与特定c-血红素的还原无关，也不能模拟为能斯特单电子氧化还原中心。相反，740 nm光谱带出现的电位范围取决于滴定是在氧化还是还原方向进行。一种可能的解释是，740 nm光谱带是氧化态高自旋P460血红素的特性，而不是低自旋态的特性，并且两种自旋态之间的转变根据电化学滴定的方向在不同电位下发生。
