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A totally synthetic histidine-2 ferredoxin: thermal stability and redox properties

Smith, Eugene T. and Tomich, John M. and Iwamoto, Takeo and Richards, John H. and Mao, Yuan and Feinberg, Benjamin A. (1991) A totally synthetic histidine-2 ferredoxin: thermal stability and redox properties. Biochemistry, 30 (50). pp. 11669-11676. ISSN 0006-2960. doi:10.1021/bi00114a009.

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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 Å away.

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Additional Information:© 1991 American Chemical Society. Received May 28, 1991; Revised Manuscript Received September 10, 1991. We thank Dr. D. W. Bennett and Dr. D. G. Nettesheim Department of Chemistry, University of Wisconsin- Milwaukee) for their assistance in the electrostatic potential calculations, C. M. Gorst (Department of Chemistry, University of Wisconsin-Milwaukee) for her assistance in obtaining EPR spectra, Dr. J. E. Wampler (Department of Biochemistry, University of Georgia) for generating the stereo image in Figure 5, and Dr. L. Noodleman (Scripps Research Clinic, La Jolla, CA) for providing the calculated iron-sulfur cluster charge sets prior to publication. This research was supported by a grant to B.A.F. from the National Institutes of Health (GM41927-01) and with funds from the University of Wisconsin-Milwaukee Graduate School.
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Issue or Number:50
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Official Citation:Smith, E. T., Tomich, J. M., Iwamoto, T., Richards, J. H., Mao, Y., & Feinberg, B. A. (1991). A totally synthetic histidine-2 ferredoxin: thermal stability and redox properties. Biochemistry, 30(50), 11669-11676. doi: 10.1021/bi00114a009
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ID Code:57157
Deposited On:01 May 2015 21:07
Last Modified:10 Nov 2021 21:08

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