Luykx D M, Duine J A, de Vries S
Kluyver Institute of Biotechnology, Delft University of Technology, The Netherlands.
Biochemistry. 1998 Aug 11;37(32):11366-75. doi: 10.1021/bi972972y.
The EPR spectra of three different molybdoprotein aldehyde dehydrogenases, one purified from Comamonas testosteroni and two purified from Amycolatopsis methanolica, showed in their oxidized state a novel type of signal. These three enzymes contain two different [2Fe-2S] centers, one flavin and one molybdopterin cytosine dinucleotide, as cofactors all of which are expected to be EPR silent in the oxidized state. The new EPR signal is isotropic with g = 2.004 both at X-band and Q-band frequencies, consists of six partially resolved lines, and shows Curie temperature behavior suggesting that the signal is due to an organic radical with S = 1/2. The EPR spectra of Comamonas testosteroni aldehyde dehydrogenase obtained after cultivation in media containing 15NH4Cl and/or after substitution of H2O for D2O show the presence of both nitrogen and proton hyperfine interactions. Simulations of the spectra of the four possible isotope combinations yield a single set of hyperfine coupling constants. The electron spin shows hyperfine interaction with a single I = 1 (0.9 mT) ascribed to a N nucleus, with a single I = 1/2 (1.5 mT) ascribed to one nonexchangeable H nucleus, and with two, exchangeable, identical I = 1/2 spins (0.6 mT) ascribed to two identical exchangeable protons. Taken together, the observations and simulations rule out amino acid residues or flavin as the origin of the radical. The values of the various hyperfine coupling constants are consistent with the properties expected for a molybdenum(VI)-trihydropterin radical in which the N5 atom is engaged in two hydrogen-bonding interactions with the protein. The majority of the electron (spin) density of the radical is located at and around the N5 atom and at the proton bound to the C6 atom of the pterin ring. The EPR spectrum of the molybdopterin radical broadens above 65 K and is no longer detectable above 168 K, indicating that it is not magnetically isolated. The line broadening is ascribed to cross-relaxation with a nearby, rapidly relaxing, oxidized [2Fe-2S] center involving its magnetic S = 1 excited state in this process. The amount of radical was apparently not changed by addition of aldehydes or oxidants, but it disappeared upon reduction by sodium dithionite. Therefore, whether the molybdenum(VI) trihydropterin radical as detected here is a functional intermediate in catalysis remains to be investigated further.
三种不同的钼辅因子蛋白醛脱氢酶的电子顺磁共振（EPR）谱显示，一种从睾丸丛毛单胞菌（Comamonas testosteroni）纯化得到，另外两种从甲醇拟无枝酸菌（Amycolatopsis methanolica）纯化得到，在其氧化态下呈现出一种新型信号。这三种酶含有两种不同的[2Fe-2S]中心、一个黄素和一个钼蝶呤胞嘧啶二核苷酸作为辅因子，所有这些在氧化态下预计都是EPR沉默的。新的EPR信号在X波段和Q波段频率下都是各向同性的，g = 2.004，由六条部分分辨的谱线组成，并表现出居里温度行为，表明该信号是由一个S = 1/2的有机自由基引起的。在含有15NH4Cl的培养基中培养后和/或用D2O替代H2O后获得的睾丸丛毛单胞菌醛脱氢酶的EPR谱显示存在氮和质子超精细相互作用。对四种可能的同位素组合的谱线进行模拟得到了一组单一的超精细耦合常数。电子自旋与一个归因于N原子核的单一I = 1（0.9 mT）、一个归因于一个非交换性H原子核的单一I = 1/2（1.5 mT）以及两个归因于两个相同可交换质子的可交换、相同的I = 1/2自旋（0.6 mT）表现出超精细相互作用。综合来看，这些观察结果和模拟排除了氨基酸残基或黄素作为自由基的来源。各种超精细耦合常数的值与预期的钼（VI）-三氢蝶呤自由基的性质一致，其中N5原子与蛋白质参与两个氢键相互作用。自由基的大部分电子（自旋）密度位于N5原子及其周围以及与蝶呤环C6原子结合的质子处。钼蝶呤自由基的EPR谱在65 K以上变宽，在168 K以上不再可检测到，表明它不是磁隔离的。谱线变宽归因于与附近快速弛豫的氧化[2Fe-2S]中心的交叉弛豫，在此过程中涉及其磁S = 1激发态。添加醛或氧化剂后自由基的量显然没有变化，但用连二亚硫酸钠还原后它消失了。因此，这里检测到的钼（VI）三氢蝶呤自由基是否是催化过程中的功能中间体还有待进一步研究。
