Structure of an electron transfer complex. I. Covalent cross-linking of cytochrome c peroxidase and cytochrome c.

B Waldmeyer,H R Bosshard

doi:10.1016/s0021-9258(18)89197-9

Abstract

Cytochrome c peroxidase and cytochrome c form a noncovalent electron transfer complex in the course of the peroxidase-catalyzed reduction of H2O2. The two hemoproteins were cross-linked in 40% yield to a covalent 1:1 complex with the aid of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide. The covalent complex was found to be a valid model of the noncovalent electron transfer complex for the following reasons. The covalent complex had only 5% residual peroxidase activity toward exogeneous ferrocytochrome c indicating that the cross-linked cytochrome c covers the electron-accepting site of cytochrome c peroxidase. The residual peroxidase activity was almost independent of ionic strength indicating that the electron-accepting site is much less accessible even when ionic bonds between the two cross-linked hemoproteins are severed. The rate of reduction of heme c by ascorbate is 15 times slower in the covalent complex than in free cytochrome c and is independent of ionic strength. Although the covalent complex may not have been entirely pure with respect to the number and location of the cross-links, two major cross-links could be localized to within a few residues. One is from Lys 13 of cytochrome c to an acidic residue in positions 32, 33, 34, 35, or 37 of cytochrome c peroxidase, the other from Lys 86 of cytochrome c to a carboxyl group in the same cluster of acidic residues. The result stresses the importance of a peculiar stretch of acidic residues of cytochrome c peroxidase and of Lys 13 and 86 of cytochrome c.

Highlights

Barbara Waldmeyer and HansRudolf Bosshard$ From the Biochemisches Znstitut der Uniuersitat Zurich, Winterthurerstrass1e90, CH-8057 Zurich, Switzerland
Some support has come from comparison of primary structuresand fromchemicalmodification of amino acidside of ionic strength indicating that the electron-acceptingchains of hemoproteins
Synthesis and Purification of Covalent Complex-We have shown before that cytochrome c is covalently cross-linked to cytochrome c peroxidase by treatment with the water-soluble carbodiimide EDC [21]

Summary

COVALENTCROSS-LINKINGOFCYTOCHROME c PEROXIDASEAND CYTOCHROME c*

Barbara Waldmeyer and HansRudolf Bosshard$ From the Biochemisches Znstitut der Uniuersitat Zurich, Winterthurerstrass1e90, CH-8057 Zurich, Switzerland. Noncovalent electron transfercomplex in the courseof Closest to an actual crystal structcuorme es afitting procedure the peroxidase-catalyzed reduction of HzOz. The two wherebyindividual crystal structures are brought together, hemoproteins were cross-linked in 40%yield to a covalent 1:l complex withtheaid of l-ethyl-3-(3-dimethylaminopropy1)carbodiimide. Re- chrome c peroxidase-cytochrome c complex is a valid model cent progress on heme enzyme structure and function indi- for the cytochrome oxidase-cytochrome c complex which is catesthatquiteoftentheelectronhasto overcome large less well amenable to the sorot f analysis presentedhere. The question arises Inthispaper we reportourresults on cross-linking of astowhatextentfunctional groups of the protein or the the cytochrome c peroxidase-cytochrome c complex by the polypeptide backbone are involved in the electron transfer water-soluble carbodiimide EDC.’. For a satisfactoryanswer we need to knowmore formation of a covalent 1:1 complex by treatment of cytoabout the intermolecular interface of electron transfer com- chrome c peroxidase and cytochrome c with dithiobis(sucplexes. Results from the two experimental strategies are complementary and invery good agreement

RESULTS

30 L-R-A E-DD-E-Y-D-N-Y-I-G-Y-G-P-V-L-V-R

DISCUSSION