A totally synthetic histidine-2 ferredoxin: thermal stability and redox properties.
The entire polypeptide of Clostridium pasteurianum ferredoxin (Fd) with a site-substituted tyrosine-2----histidine-2 was synthesized using standard t-Boc procedures, reconstituted to the 2[4Fe-4S] holoprotein, and compared to synthetic C. pasteurianum and native Fds. Although histidine-2 is commonly found in thermostable clostridial Fds, the histidine-2 substitution into synthetic C. pasteurianum Fd did not significantly increase its thermostability. The reduction potential of synthetic histidine-2 Fd was -343 and -394 mV at pH 6.4 and 8.7, respectively, versus standard hydrogen electrode. Similarly, Clostridium thermosaccharolyticum Fd which naturally contains histidine-2 was previously determined to have a pH-dependent reduction potential [Smith, E.T., & Feinberg, B.A. (1990) J. Biol. Chem. 265, 14371-14376]. An electrostatic model was used to calculate the observed change in reduction potential with pH for a homologous ferredoxin with a known X-ray crystal structure containing a hypothetical histidine-2. In contrast, the reduction potential of both native C. pasteurianum Fd and synthetic Fd with the C. pasteurianum sequence was -400 mV versus standard hydrogen electrode and was pH-independent [Smith, E.T., Feinberg, B.A., Richards, J.H., & Tomich, J.M. (1991) J. Am. Chem. Soc. 113, 688-689]. On the basis of the above results, we conclude that the observed pH-dependent reduction potential for both synthetic and native ferredoxins that contain histidine-2 is attributable to the electrostatic interaction between histidine-2 and iron-sulfur cluster II which is approximately 6 A away.
Smith
E T
ET
Department of Chemistry, University of Wisconsin-Milwaukee 53201.
Tomich
J M
JM
Iwamoto
T
T
Richards
J H
JH
Mao
Y
Y
Feinberg
B A
BA
D000595:Amino Acid Sequence; D002851:Chromatography, High Pressure Liquid; D003013:Clostridium; D004563:Electrochemistry; D004578:Electron Spin Resonance Spectroscopy; D005288:Ferredoxins / Q000138:chemical synthesis* / Q000737:chemistry; D006358:Hot Temperature; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D013057:Spectrum Analysis
D005288:Ferredoxins; C072056:histidine-2 ferredoxin