Comparative Structural-Functional Characterization of Recombinant and Natural Adrenodoxin. Interaction with Cytochrome P450scc

G. I. Lepesheva, T. N. Azeva, N. V. Strushkevich, T. B. Adamovich, T. S. Cherkesova, S. A. Usanov

Research output: Contribution to journalArticlepeer-review

4 Citations (Scopus)

Abstract

The conditions for heterologous expression of recombinant bovine adrenodoxin in E. coli have been optimized, thus reaching expression levels up to 12-14 μmoles per liter of culture medium. A highly efficient method for purification of this recombinant ferredoxin from the E. coli cells has been developed. The structural-functional properties of the highly purified recombinant protein have been characterized and compared to those of natural adrenodoxin purified from bovine adrenocortical mitochondria. In contrast to natural adrenodoxin, which is characterized by microheterogeneity, the recombinant adrenodoxin is homogeneous as judged by N- and C-terminal amino acid sequencing, and its sequence corresponds to the full-length mature form of adrenodoxin containing 128 amino acid residues. The interactions of the natural and recombinant adrenodoxins with cytochrome P450scc have been studied and compared with respect to: the efficiency of their enzymatic reduction of cytochrome P450scc in a reconstituted system; the ability of the immobilized adrenodoxins to bind cytochrome P450scc; the ability of the adrenodoxins to induce a spectral shift of cytochrome P450scc and to effect the average polarity of exposed tyrosines in the low-spin cytochrome P450scc. The recombinant adrenodoxin is functionally active and in the reduced state as well as at low ionic strength it displays higher affinity to cytochrome P450scc as compared to the natural bovine adrenocortical adrenodoxin. The possible role of the C-terminal sequence of the adrenodoxin molecule in its interaction with cytochrome P450scc as well as the advantages of using the recombinant protein instead of the natural one are discussed.

Original languageEnglish
Pages (from-to)1079-1088
Number of pages10
JournalBiochemistry (Moscow)
Volume64
Issue number9
Publication statusPublished - Sep 1999
Externally publishedYes

Keywords

  • Adrenodoxin
  • Conformational dynamics
  • Cytochrome p450scc
  • Heterologous expression
  • Protein-protein interaction

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