An Alternative Proposal for the Reaction Mechanism of Light-Dependent Protochlorophyllide Oxidoreductase

Light-dependent
protochlorophyllide oxidoreductase is one of the
few known enzymes that require a quantum of light to start their catalytic
cycle. Upon excitation, it uses NADPH to reduce the C17–C18 in its substrate (protochlorophyllide) through
a complex mechanism that has heretofore eluded precise determination.
Isotopic labeling experiments have shown that the hydride-transfer
step is very fast, with a small barrier close to 9 kcal mol–1, and is followed by a proton-transfer step, which has been postulated
to be the protonation of the product by the strictly conserved Tyr189
residue. Since the structure of the enzyme–substrate complex
has not yet been experimentally determined, we first used modeling
techniques to discover the actual substrate binding mode. Two possible
binding modes were found, both yielding stable binding (as ascertained
through molecular dynamics simulations) but only one of which placed
the critical C17C18 bond consistently close to the NADPH pro-S hydrogen and to Tyr189. This binding
pose was then used as a starting point for the testing of previous
mechanistic proposals using time-dependent density functional theory.
The quantum-chemical computations clearly showed that such mechanisms
have prohibitively high activation energies. Instead, these computations
showed the feasibility of an alternative mechanism initiated by excited-state
electron transfer from the key Tyr189 to the substrate. This mechanism
appears to agree with the extant experimental data and reinterprets
the final protonation step as a proton transfer to the active site
itself rather than to the product, aiming at regenerating it for another
round of catalysis.

依赖光的原叶绿素酸酯氧化还原酶（Light-dependent protochlorophyllide oxidoreductase）是目前已知少数需要光量子以启动催化循环的酶之一。经激发后，该酶利用烟酰胺腺嘌呤二核苷酸磷酸（NADPH）还原底物（原叶绿素酸酯）上的C17-C18双键，其催化机制复杂且迄今尚未被精准阐明。同位素标记实验证实，氢化物转移步骤速率极快，活化能垒仅约9 kcal mol–1，随后紧随质子转移步骤；该步骤曾被推测为由高度保守的Tyr189残基将质子转移至产物。由于该酶-底物复合物的结构尚未通过实验解析，我们首先采用建模技术以探明底物的真实结合模式。共发现两种可行的结合模式，二者均能形成稳定结合（经分子动力学模拟验证），但仅其中一种可使关键的C17=C18双键始终处于靠近NADPH的pro-S氢原子以及Tyr189残基的位置。随后，以该结合构象为起点，利用含时密度泛函理论（time-dependent density functional theory）对此前提出的多种机制假说进行验证。量子化学计算结果清晰表明，此类机制的活化能高得难以实现。相反，计算结果证实了一种替代机制的可行性：该机制由激发态下关键残基Tyr189向底物发生电子转移所启动。该机制与现有实验数据相符，且将最终的质子化步骤重新阐释为：质子向活性中心自身转移，而非向产物转移，其目的是为新一轮催化循环再生该关键残基。