Gorren A C, de Vries S, Duine J A
Department of Microbiology and Enzymology, Delft University of Technology, The Netherlands.
Biochemistry. 1995 Aug 1;34(30):9748-54. doi: 10.1021/bi00030a012.
The binding of monovalent cations to methylamine dehydrogenase in the semiquinone state (MADHsq) at a site close to the tryptophan tryptophylquinone (TTQ) active center is demonstrated in experiments which show that the radical EPR signal of MADHsq is considerably broadened in the presence of Cs+, NH4+, and, to a smaller extent, Na+. The cations also stabilize the semiquinone state, as is evident from the increase of the EPR intensity they induce. On the basis of the optical absorbance spectra, two slightly different forms of MADHsq can be discerned. One form, with the main band at 425 nm, is observed at low pH and in the presence of NH4+, whereas the other, with the main band at 429 nm, is observed at high pH and in the presence of Cs+ or Na+. Stopped-flow studies of the oxidation by amicyanin of MADHred via MADHsq to MADHox show a strong stimulation of the first step by monovalent cations. It is shown that it is primarily the actual electron transfer rate, rather than the affinity of MADHred for amicyanin, that is affected by cations. Values for the dissociation constants of the monovalent cations for MADHred, estimated from the kinetic experiments, are higher than those that were previously determined for MADHox, and can be deduced to be higher than those for MADHsq as well. The results are discussed within the context of the electron transfer theory.
实验表明，单价阳离子在靠近色氨酸-色氨醌（TTQ）活性中心的位点与处于半醌态的甲胺脱氢酶（MADHsq）结合。这些实验显示，在Cs⁺、NH₄⁺存在时，以及在较小程度上在Na⁺存在时，MADHsq的自由基电子顺磁共振（EPR）信号会显著变宽。这些阳离子还能稳定半醌态，这从它们诱导的EPR强度增加中可以明显看出。根据光吸收光谱，可以辨别出两种略有不同形式的MADHsq。一种形式的主峰在425nm，在低pH值和NH₄⁺存在时观察到；而另一种形式的主峰在429nm，在高pH值和Cs⁺或Na⁺存在时观察到。通过MADHsq将MADHred氧化为MADHox的过程中，对氨蓝蛋白氧化MADHred的停流研究表明，单价阳离子对第一步反应有强烈的促进作用。结果表明，主要是实际的电子转移速率，而不是MADHred对氨蓝蛋白的亲和力，受到阳离子的影响。从动力学实验估计的单价阳离子对MADHred的解离常数的值，高于先前测定的MADHox的解离常数的值，并且可以推断也高于MADHsq的解离常数的值。在电子转移理论的背景下对这些结果进行了讨论。
