Rational design of a mononuclear metal site into the archaeal Rieske-type protein scaffold.
Proteins containing Rieske-type [2Fe-2S] clusters play essential functions in all three domains of life. We engineered the two histidine ligands to the Rieske-type [2Fe-2S] cluster in the hyperthermophilic archaeal Rieske-type ferredoxin from Sulfolobus solfataricus to modify types and spacing of ligands and successfully converted the metal and cluster type at the redox-active site with a minimal structural change to a native Rieske-type protein scaffold. Spectroscopic analyses unambiguously established a rubredoxin-type mononuclear Fe3+/2+ center at the engineered local metal-binding site (Zn2+ occupies the iron site depending on the expression conditions). These results show the importance of types and spacing of ligands in the in vivo cluster recognition/insertion/assembly in biological metallosulfur protein scaffolds. We suggest that early ligand substitution and displacement events at the local metal-binding site(s) might have primarily allowed the metal and cluster type conversion in ancestral redox protein modules, which greatly enhanced their capabilities of conducting a wide range of unique redox chemistry in biological electron transfer conduits, using a limited number of basic protein scaffolds.
Iwasaki
Toshio
T
Department of Biochemistry and Molecular Biology, Nippon Medical School, Sendagi, Bunkyo-ku, Tokyo 113-8602, Japan. tiwasaki@nms.ac.jp
Kounosu
Asako
A
Tao
Ye
Y
Li
Zhongrui
Z
Shokes
Jacob E
JE
Cosper
Nathaniel J
NJ
Imai
Takeo
T
Urushiyama
Akio
A
Scott
Robert A
RA
D000595:Amino Acid Sequence; D000818:Animals; D019843:Archaeal Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites; D002417:Cattle; D014450:Electron Transport Complex III / Q000737:chemistry / Q000378:metabolism*; D005288:Ferredoxins / Q000378:metabolism*; D007501:Iron / Q000378:metabolism; D007506:Iron-Sulfur Proteins / Q000737:chemistry / Q000378:metabolism*; D008024:Ligands; D008670:Metals / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016366:Open Reading Frames; D010084:Oxidation-Reduction; D011487:Protein Conformation; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D016944:Sulfolobus / Q000378:metabolism*; D015032:Zinc / Q000378:metabolism
D019843:Archaeal Proteins; D005288:Ferredoxins; D007506:Iron-Sulfur Proteins; D008024:Ligands; D008670:Metals; C037989:Rieske iron-sulfur protein; D007501:Iron; D014450:Electron Transport Complex III; D015032:Zinc