414 IRON-CONTAINING PROTEINS AND ENZYMES
formational changes in the two oxidation states, around the met80 ligand and
in the heme (especially around the pyrrole A ring and its attached propionate
oxygens).
The Brayer group next began a series of mutational studies on yeast iso - 1
ferro - and ferricytochromes c. The fi rst mutant replaced the tyrosine residue
at position 67 with phenylalanine — a Y67F mutant. Both oxidation states were
studied: (1) the Fe(II) – Y67F mutant deposited as PDB: 1CTZ and (2) the
Fe(III) – Y67F mutant as PDB: 1CTY.^112 The reduced state Y67F mutant
appears to be very similar to its wild - type (WT) counterpart. Differences are
seen for the oxidized mutant (PDB: 1CTY) in comparison to its WT counter-
part (PDB: 2YCC). While little change is seen in overall polypeptide chain
positioning for either oxidation state, more substantial changes in thermal
factor (and therefore chain mobility) are seen, especially comparing the oxi-
dized wild - type and Y67F mutant proteins. In the oxidized Y67F mutant, the
heme porphyrin ligand is slightly less distorted from planarity — PDB: 2YCC
average angular deviation of pyrrole rings from the pyrrole nitrogens ’ plane
= 12.2 ° , PDB: 1CTY average angular deviation = 11.6 °. Table 2 of reference
112 shows WT versus Y67F mutant comparisons of heme geometry — angular
deviations for heme and axial ligands plus heme iron – ligand bond distances.
The oxidized Y67F mutant heme met80 ligand changes its orientation from
the wild - type; for instance, the C ε methyl group moves ∼ 1.8 Å and rotates ∼ 40 °
about the ligand bond. This movement may be connected to the observation
that the met80 - conformation - stabilizing wat166 molecule present in the WT
protein is missing in the Y67F mutant. Additionally, the stabilizing hydrogen
bond between met80 S δ and tyr67 OH group is not possible for the Y67F
mutant. The heme propionate group (O1A and O2A) attached to pyrrole ring
A has changed orientation in WT versus Y67F structures as have the orienta-
tions of conserved arg38 and asn52 side chains. These reorientations lead to
changes in hydrogen - bonding patterns in this region of WT versus Y67F
mutant proteins. The differences between both reduced and oxidized wild - type
and mutant forms are collected in Table 7 of reference 112. The differences
noted include: (1) a drop in the midpoint reduction potential for the Y67F
mutant (234 mV) compared to that for the WT (290 mV); (2) changes in ther-
modynamic properties; and (3) changes in UV – visible absorption spectra that
indicate perturbation of theπ - conjugated heme system in the mutant protein.
The conclusion must be that while the overall fold of the cytochrome c protein
does not change, mutations near the heme site will signifi cantly alter the
heme environment and thus alter its electron transfer behavior with redox
partners.
The Brayer group continued its X - ray crystallographic structural study of
yeast iso - 1 - cytochrome c variants with the following: (1) PDB: 1CRG, Fe(III) –
asparagine52 to isoleucine52 (N52I) mutant; (2) PDB: 1CRH, Fe(II) – N52I
mutant; (3) PDB: 1CRI, Fe(III) – N52I - Y67F mutant; and (4) PDB: 1CRJ,
Fe(II) – N52I - Y67F mutant.^113 In all mutants, the most prominent structural
difference from wild - type enzyme is the displacement of the conserved wat166