bi201344h_si_001.pdf (3.48 MB)
Zinc-Substituted Cytochrome P450cam: Characterization of Protein Conformers F420 and F450 by Photoinduced Electron Transfer
journal contribution
posted on 2012-02-21, 00:00 authored by Katarzyna
I. Jankowska, Cynthia V. Pagba, Piotr PiotrowiakMetal substitution of heme proteins is widely applied
in the study
of biologically relevant electron transfer (ET) reactions. It has
been shown that many modified proteins remain in their native conformation
and can provide useful insights into the molecular mechanism of electron
transfer between the native protein and its substrates. We investigated
ET reactions between zinc-substituted cytochrome P450cam and small organic compounds such as quinones and ferrocene, which
are capable of accessing the protein’s hydrophobic channel
and binding close to the active site, like its native substrate, camphor.
Following the substitution method developed by Gunsalus and co-workers
[Wagner, G. C., et al. (1981) J. Biol. Chem. 256,
6262–6265], we have identified two dominant forms of the zinc-substituted
protein, F450 and F420, that exhibit different photophysical and photochemical
properties. The ET behavior of F420 suggests that hydrophobic redox-active
ligands are able to penetrate the hydrophobic channel and place themselves
in the direct vicinity of the Zn-porphyrin. In contrast, the slower
ET quenching rates observed in the case of F450 indicate that the
association is weak and occurs outside of the protein channel. Therefore,
we conclude that F420 corresponds to the open structure of the native
cytochrome P450cam while F450 has a closed or partially
closed channel that is characteristic of the camphor-containing cytochrome
P450cam. The existence of two distinct conformers of Zn-bound
P450cam is consistent with the findings of Goodin and co-workers
[Lee, Y.-T., et al. (2010) Biochemistry 49, 3412–3419]
and has significant consequences for future electron transfer studies
on this popular metalloenzyme.