426 IRON-CONTAINING PROTEINS AND ENZYMES
mutant cytochrome c (cyto c) (PDB: 1S6V, resolution 1.88 Å ).^131 The higher
resolution of this crystal structure identifi ed the positions of ordered water
molecules that bridged the two proteins through hydrogen bonds. As with the
PDB: 2PCC structure, the direct electrostatic interactions do not appear critical
in complex formation. The crosslinked CcP - cyto c complex behaves normally.
UV - visible spectroscopic and stopped - fl ow kinetic studies indicate that the CcP
domain of the crosslinked complex reacts with H 2 O 2 to produce the so - called
compound I (see description of CcP above). Stopped fl ow studies also indicate
that intramolecular electron transfer between the crosslinked ferrocytochrome
c and the CcP trp191 cation radical occurs within two milliseconds. The refer-
ence 131 authors believe these results indicate that the ferrocytochrome c
resides at or close to its physiological position in the PDB: 1S6V covalent
complex (called PCXL in reference 131 ). Further evidence is shown in kinetic
experiments using exogenously added yeast and horse cytochrome c. While
wild - type CcP shows high and fast steady - state and single - turnover activity
with exogenous cytochromes c, the PCXL covalently bonded CCP – cyto c
complex shows much less activity and reacts much more slowly. The authors
submit these experiments to indicate that CcP does not have a second inde-
pendent site for physiologically relevant electron transfer.
Substitution of zinc(II) ions into cytochrome c peroxidase (ZnCcP) has
been used to exploit photoactivation of electron transfer (eT) reactions since
the mid - 1990s.^132 The ZnCcP triplet state (^3 ZnCcP) reduces Fe(III) cytochrome
c, and then back electron transfer recombines the charge separation to com-
plete the catalytic cycle (see Figure 7.36 ).
In the experiment,^3 ZnCcP is produced by laser photolysis, then transient
absorption spectroscopy follows the formation ( ke ) and decay ( keb ) of ZnCcP • +
in wild - type and mutant crystals. Kang and Crane have studied the effects
of interface mutations on electron transfer rates in single crystals using
complexes between a zinc - substituted cytochrome c peroxidase (ZnCcP) and
site - directed mutants of yeast cytochrome c (yCc). The mutants replaced the
Figure 7.36 3 ZnCcP – Fe(III) reaction cycle. (Adapted with permission from Figure 1
of reference 133. Copyright 2005 National Academy of Sciences, U.S.A.)
[^3 Zn(II)CcP:Fe(III)cyto c[[Zn(II)CcP:Fe(III)cyto c[kd hν [Zn(II)CcP.+:Fe(II)cyto c[ [[Zn(II)CcP(W191.+):Fe(II)cyto ckekeb(structures may be analogous
to compounds I or II intermediates
in cytochrome c peroxidase
catalytic cycle)