Robbins John M, Geng Jiafeng, Barry Bridgette A, Gadda Giovanni, Bommarius Andreas S
School of Chemical and Biomolecular Engineering , Georgia Institute of Technology , Atlanta , Georgia 30332-0100 , United States.
Engineered Biosystems Building (EBB) , Georgia Institute of Technology , Atlanta , Georgia 30332-2000 , United States.
Biochemistry. 2018 Oct 9;57(40):5818-5826. doi: 10.1021/acs.biochem.8b00571. Epub 2018 Sep 27.
Formate oxidase (FOX) was previously shown to contain a noncovalently bound 8-formyl FAD (8-fFAD) cofactor. However, both the absorption spectra and the kinetic parameters previously reported for FOX are inconsistent with more recent reports. The ultraviolet-visible (UV-vis) absorption spectrum reported in early studies closely resembles the spectra observed for protein-bound 8-formyl flavin semiquinone species, thus suggesting FOX may be photosensitive. Therefore, the properties of dark and light-exposed FOX were investigated using steady-state kinetics and site-directed mutagenesis analysis along with inductively coupled plasma optical emission spectroscopy, UV-vis absorption spectroscopy, circular dichroism spectroscopy, liquid chromatography and mass spectrometry, and electron paramagnetic resonance (EPR) spectroscopy. Surprisingly, these experimental results demonstrate that FOX is deactivated in the presence of light through generation of an oxygen stable, anionic (red) 8-fFAD semiquinone radical capable of persisting either in an aerobic environment for multiple weeks or in the presence of a strong reducing agent like sodium dithionite. Herein, we study the photoinduced formation of the 8-fFAD semiquinone radical in FOX and report the first EPR spectrum of this radical species. The stability of the 8-fFAD semiquinone radical suggests FOX to be a model enzyme for probing the structural and mechanistic features involved in stabilizing flavin semiquinone radicals. It is likely that the photoinduced formation of a stable 8-fFAD semiquinone radical is a defining characteristic of 8-formyl flavin-dependent enzymes. Additionally, a better understanding of the radical stabilization process may yield a FOX enzyme with more robust activity and broader industrial usefulness.
甲酸氧化酶（FOX）先前已被证明含有一种非共价结合的8-甲酰基黄素腺嘌呤二核苷酸（8-fFAD）辅因子。然而，先前报道的FOX的吸收光谱和动力学参数与最近的报道不一致。早期研究中报道的紫外可见（UV-vis）吸收光谱与蛋白质结合的8-甲酰基黄素半醌物种所观察到的光谱非常相似，因此表明FOX可能对光敏感。因此，利用稳态动力学和定点诱变分析，结合电感耦合等离子体发射光谱、紫外可见吸收光谱、圆二色光谱、液相色谱和质谱以及电子顺磁共振（EPR）光谱，对黑暗和光照下的FOX的性质进行了研究。令人惊讶的是，这些实验结果表明，在光照下，FOX会通过生成一种氧稳定的阴离子（红色）8-fFAD半醌自由基而失活，该自由基能够在有氧环境中持续存在数周，或者在连二亚硫酸钠等强还原剂存在的情况下持续存在。在此，我们研究了FOX中8-fFAD半醌自由基的光诱导形成，并报道了该自由基物种的首个EPR光谱。8-fFAD半醌自由基的稳定性表明FOX是一种用于探究稳定黄素半醌自由基所涉及的结构和机制特征的模型酶。8-甲酰基黄素依赖性酶的一个决定性特征可能是光诱导形成稳定的8-fFAD半醌自由基。此外，对自由基稳定过程的更好理解可能会产生一种具有更强活性和更广泛工业用途的FOX酶。
