Resonance Raman spectroscopic analysis of the iron-sulfur cluster redox chain of the Ralstonia eutropha membrane-bound [NiFe]-hydrogenase

被引:6
作者
Siebert, Elisabeth [1 ]
Schmidt, Andrea [2 ]
Frielingsdorf, Stefan [1 ]
Kalms, Jacqueline [2 ,3 ]
Kuhlmann, Uwe [1 ]
Lenz, Oliver [1 ]
Scheerer, Patrick [2 ]
Zebger, Ingo [1 ]
Hildebrandt, Peter [1 ]
机构
[1] Tech Univ Berlin, Inst Chem, Sekr PC14,Str 17 Juni 135, D-10623 Berlin, Germany
[2] Charite Univ Med Berlin, Humboldt Univ Berlin, Free Univ Berlin, Inst Med Phys & Biophys CC2, Charitepl 1, D-10117 Berlin, Germany
[3] Univ Leicester, Leicester Inst Struct & Chem Biol, Leicester, Leics, England
关键词
electron transfer; hydrogenase; iron-sulfur cluster; protein crystals; Raman spectroscopy; 4FE-3S CLUSTER; ESCHERICHIA-COLI; ACTIVE-SITE; O-2-TOLERANT; OXYGEN; HYDROGENASE; PROTEINS; 3FE-4S;
D O I
10.1002/jrs.6163
中图分类号
O433 [光谱学];
学科分类号
0703 ; 070302 ;
摘要
Iron-sulfur (Fe-S) centers are versatile building blocks in biological electron transfer chains because their redox potentials may cover a wide potential range depending on the type of the cluster and the specific protein environment. Resonance Raman (RR) spectroscopy is widely used to analyze structural properties of such cofactors, but it remains still a challenge to disentangle the overlapping signals of metalloproteins carrying several Fe-S centers. In this work, we combined RR spectroscopy with protein engineering and X-ray crystallography to address this issue on the basis of the oxygen-tolerant membrane-bound hydrogenase from Ralstonia eutropha that catalyzes the reversible conversion of hydrogen into protons and electrons. Besides the NiFe-active site, this enzyme harbors three different Fe-S clusters constituting an electron relay with a distal [4Fe-4S], a medial [3Fe-4S], and an unusual proximal [4Fe-3S] cluster that may carry a hydroxyl ligand in the superoxidized state. RR spectra were measured from protein crystals by varying the crystal orientation with respect to the electric field vector of the incident laser to achieve a preferential RR enhancement for individual Fe-S clusters. In addition to spectral discrimination by selective reduction of the proximal cluster, protein engineering allowed for transforming the proximal and medial cluster into standard cubane-type [4Fe-4S] centers in the C19G/C120G and P242C variants, respectively. The latter variant was structurally characterized for the first time in this work. Altogether, the entirety of the RR data provided the basis for identifying the vibrational modes characteristic of the various cluster states in this "model" enzyme as a prerequisite for future studies of complex (FeS)-based electron transfer chains.
引用
收藏
页码:2621 / 2632
页数:12
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