CYTOCHROMES c 415
molecule. As described previously, this same water molecule moves its position
substantially, depending on the oxidation state of the enzyme (see discussion
of PDB: 1YCC and PDB: 2YCC above). The reference 113 authors suggest
that the importance of wat166, along with its associated hydrogen - bonding
network, rests on three major attributes: (1) wat166 ’ s position modifi es
the hydrogen - bonding network surrounding met80 in an oxidation - state -
dependent manner; (2) wat166 maintains the spatial relationships between
nearby amino acid side chains and therefore establishes the hydrogen - bonding
network in this region of the protein (especially regarding the tyr67 hydroxyl
group ’ s hydrogen bond to S δ of iron axial ligand met80); and (3) wat166
appears to mediate increases in the mobility of three nearby peptide segment
in the enzyme ’ s oxidized state. Regarding point (2) above, the reference 113
authors believe that the absence or presence of the tyr67 OH to met80 ’ s S δ
hydrogen bond affects the electron - withdrawing power of the met80 ligand
and therefore is a factor in controlling the midpoint reduction potential in this
cytochrome c (see discussion of PDB: 1CTY structure above). Regarding point
(3) above, their research shows that, in the absence of wat166, mobility changes
based on oxidation state do not occur in the N52I or N52I - Y67F mutants. This
is an important fi nding since it is thought that changes in protein side - chain
mobility with oxidation state are related to oxidation - state - dependent interac-
tions between cytochrome c and its redox partners. The reference 113 authors
also conclude that movement of amino acid side chains (particularly asn52)
that participate in the hydrogen - bonding network around the heme pyrrole A
propionate are responsible for oxidation - state conformational changes in this
region.
In a 1996 Biochemistry paper, Brayer, Mauk, and co - workers published a
study of cytochrome c electron transfer reactivity in relation to mechanistic
and structural contributions by important surface and internal amino acid
residues. Measurement of midpoint reduction potentials for wild - type versus
mutant proteins showed that wild - type proteins had the highest potential at
290 mV. Mutants that changed the phe82 surface residue, a conserved aa
residue thought to be important in interactions between cytochrome c and its
redox partners, lowered the reduction potential less. However, a trend toward
lower potential tracked with size of the variant amino acid substituted for
phe82. For instance, the mutant phe82tyr (280 mV) exhibited less change than
the phe82ser (255 mV) or the phe82gly (247 mV) mutants.^114 Mutants that
varied conserved internal amino acids thought to be important in hydrogen -
bonding networks near the cytochrome c heme — thr78, tyr67, asn52 — caused
larger changes in the midpoint reduction potential — asn52ala (257 mV), tyr-
67phe (236 mV), and thr78gly (245 mV). The group also studied reduction
kinetics of wild - type and mutant ferricytochromes using [Fe(EDTA)] 2 − (EDTA
= ethylenediaminetetraacetate) as a reductant and the oxidation kinetics
of WT and mutant ferrocytochromes using [Co(phen) 3 ] 3+ (phen = 1,10 -
phenanthroline) as an oxidant. They applied Marcus theory (see Section 1.8 )
to calculate self - exchange rates ( k 11 or k 11 corr) for the studied proteins. Electron