Institut für Experimentalphysik, Freie Universität Berlin, Berlin, Germany.
Inorg Chem. 2011 Feb 7;50(3):741-8. doi: 10.1021/ic101291j. Epub 2010 Dec 29.
YedY from Escherichia coli is a new member of the sulfite oxidase family of molybdenum cofactor (Moco)-containing oxidoreductases. We investigated the atomic structure of the molybdenum site in YedY by X-ray absorption spectroscopy, in comparison to human sulfite oxidase (hSO) and to a Mo(IV) model complex. The K-edge energy was indicative of Mo(V) in YedY, in agreement with X- and Q-band electron paramagnetic resonance results, whereas the hSO protein contained Mo(VI). In YedY and hSO, molybdenum is coordinated by two sulfur ligands from the molybdopterin ligand of the Moco, one thiolate sulfur of a cysteine (average Mo-S bond length of ∼2.4 Å), and one (axial) oxo ligand (Mo═O, ∼1.7 Å). hSO contained a second oxo group at Mo as expected, but in YedY, two species in about a 1:1 ratio were found at the active site, corresponding to an equatorial Mo-OH bond (∼2.1 Å) or possibly to a shorter Mo-O(-) bond. Yet another oxygen (or nitrogen) at a ∼2.6 Å distance to Mo in YedY was identified, which could originate from a water molecule in the substrate binding cavity or from an amino acid residue close to the molybdenum site, i.e., Glu104, that is replaced by a glycine in hSO, or Asn45. The addition of the poor substrate dimethyl sulfoxide to YedY left the molybdenum coordination unchanged at high pH. In contrast, we found indications that the better substrate trimethylamine N-oxide and the substrate analogue acetone were bound at a ∼2.6 Å distance to the molybdenum, presumably replacing the equatorial oxygen ligand. These findings were used to interpret the recent crystal structure of YedY and bear implications for its catalytic mechanism.
大肠杆菌中的 YedY 是亚硫酸氧化酶家族中含钼辅酶（Moco）的氧化还原酶的新成员。我们通过 X 射线吸收光谱法研究了 YedY 中钼位的原子结构，将其与人类亚硫酸氧化酶（hSO）和 Mo(IV) 模型配合物进行了比较。K 边能量表明 YedY 中的钼为 Mo(V)，这与 X 射线和 Q 波段电子顺磁共振结果一致，而 hSO 蛋白则含有 Mo(VI)。在 YedY 和 hSO 中，钼由 Moco 中钼喋呤配体的两个硫配体、半胱氨酸的一个硫醇硫（平均 Mo-S 键长约为 2.4 Å）和一个（轴向）氧配体（Mo═O，约 1.7 Å）配位。正如预期的那样，hSO 中含有第二个氧原子，但在 YedY 中，在活性位点处发现了两种大约 1:1 的物种，它们对应于赤道 Mo-OH 键（约 2.1 Å）或可能是较短的 Mo-O(-) 键。在 YedY 中，还鉴定出了与钼距离约为 2.6 Å 的另一个氧（或氮）原子，它可能来自于底物结合腔中的水分子或靠近钼位的氨基酸残基，即 Glu104，在 hSO 中被甘氨酸取代，或 Asn45。将较差的底物二甲亚砜添加到 YedY 中，在高 pH 值下，钼的配位不变。相比之下，我们发现有迹象表明更好的底物三甲胺 N-氧化物和底物类似物丙酮与钼以约 2.6 Å 的距离结合，可能取代了赤道氧配体。这些发现被用于解释最近的 YedY 晶体结构，并对其催化机制具有启示意义。
