Stripp Sven, Sanganas Oliver, Happe Thomas, Haumann Michael
Lehrstuhl für Biochemie der Pflanzen, AG Photobiotechnologie, Ruhr-Universität Bochum, 44780 Bochum, Germany.
Biochemistry. 2009 Jun 9;48(22):5042-9. doi: 10.1021/bi900010b.
The [FeFe] hydrogenase (CrHydA1) of the green alga Chlamydomonas reinhardtii is the smallest hydrogenase known and can be considered as a "minimal unit" for biological H(2) production. Due to the absence of additional FeS clusters as found in bacterial [FeFe] hydrogenases, it was possible to specifically study its catalytic iron-sulfur cluster (H-cluster) by X-ray absorption spectroscopy (XAS) at the Fe K-edge. The XAS analysis revealed that the CrHydA1 H-cluster consists of a [4Fe4S] cluster and a diiron site, 2Fe(H), which both are similar to their crystallographically characterized bacterial counterparts. Determination of the individual Fe-Fe distances in the [4Fe4S] cluster ( approximately 2.7 A) and in the 2Fe(H) unit ( approximately 2.5 A) was achieved. Fe-C( horizontal lineO/N) and Fe-S bond lengths were in good agreement with crystallographic data on bacterial enzymes. The loss of Fe-Fe distances in protein purified under mildly oxidizing conditions indicated partial degradation of the H-cluster. Bond length alterations detected after incubation of CrHydA1 with CO and H(2) were related to structural and oxidation state changes at the catalytic Fe atoms, e.g., to the binding of an exogenous CO at 2Fe(H) in CO-inhibited enzyme. Our XAS studies pave the way for the monitoring of atomic level structural changes at the H-cluster during H(2) catalysis.
莱茵衣藻的[FeFe]氢化酶（CrHydA1）是已知最小的氢化酶，可被视为生物产氢的“最小单元”。由于其不像细菌[FeFe]氢化酶那样含有额外的铁硫簇，因此可以通过铁K边X射线吸收光谱（XAS）专门研究其催化铁硫簇（H簇）。XAS分析表明，CrHydA1 H簇由一个[4Fe4S]簇和一个双铁位点2Fe(H)组成，二者均与其晶体学表征的细菌对应物相似。实现了对[4Fe4S]簇（约2.7 Å）和2Fe(H)单元（约2.5 Å）中单个铁-铁距离的测定。铁-碳（横线O/N）和铁-硫键长与细菌酶的晶体学数据高度吻合。在轻度氧化条件下纯化的蛋白质中，铁-铁距离的损失表明H簇发生了部分降解。CrHydA1与CO和H₂孵育后检测到的键长变化与催化铁原子处的结构和氧化态变化有关，例如与CO抑制的酶中2Fe(H)处外源CO的结合有关。我们的XAS研究为监测H₂催化过程中H簇的原子水平结构变化铺平了道路。
