BIOINORGANIC CHEMISTRY A Short Course Second Edition

CYTOCHROMES c 423

while the horse Har – Co – cyt c ’ s kinetics remained biphasic (see Figure 2 of
reference 124 ). This result indicates that lysines as well as histidines can coor-
dinate to the cytochrome c metal center during refolding events in denatured
cytochrome c at neutral pH. It was also determined that his33 binds more
tightly than his26 in unfolded horse Co – cyt c, and therefore his26 mis - ligated
protein refolds 3 to 4 times faster than his33 mis - ligated protein. The conclu-
sion is that folding is limited by histidine dissociation from Co(III) in horse
Co – cyto c, and this happens faster for his26 because of the smaller loop gener-
ated when this aa residue coordinates.
Winkler, Gray, and Lyubovitsky use fl uorescence energy transfer (FET)
experiments to provide nanosecond - time - scale snapshots of the distance dis-
tributions between donor (D) and acceptor (A) labeled domains in rapidly
equilibrating populations of folding intermediates.^126 For the yeast Co – cyt c
experiments they labeled a C - terminal residue, cys102, with a dansyl fl uoro-
phore (5 - ((((2 - iodoacetyl)amino)ethyl)amino) - naphthalene - 1 - sulfonic acid),
DNS. The DNS derivatized - cys102 (donor, D) fl uoresces when the protein is
unfolded but is signifi cantly quenched by energy transfer to the Co(III) -
containing heme (acceptor, A) in the folded protein conformation. First, the
DNS(cys102) – Co cyt c is denatured by addition of guanidine hydrochloride
(GuHCl). Folding is then triggered by diluting the denatured solution in a
stopped - fl ow mixer. Dilution - mixing is synchronized with excitation by an
Nd:YAG laser (355 nm, 50 ps, 0.5 mJ) and the fl uorescence decay followed with
a picosecond streak camera. The DNS(cys102) – Co cyt c results show (1) a fast
collapse of a fraction of the DNS(cys102) – Co cyt c to a DNS – Co(III) average
distance of∼ 30 Å shortly after triggering folding; (2) the observation that most
molecules are still in an extended conformation hours after folding initiation;
(3) the formation of the Co cyt c native structure on an even longer time scale
as both extended and compact conformations collapse to the native conforma-
tion; and (4) arrival at a distance distribution characteristic of the native
protein ( ∼ 25 – 27 Å ) after about 18 hours for Co – cyt c. This is in contrast to
results for DNS(cys102) – Fe – cyto c that evolves to the native structure within
a few hundred milliseconds. The researchers also studied the behavior of these

Figure 7.35 Lysine, homoarginine and arginine.

H 2 NCHC

CH 2

OH

O

CH 2

CH 2

NH

C

NH 2

NH

H 2 NCHC

CH 2

OH

O

CH 2

CH 2

CH 2

NH 2

H 2 NCHC

CH 2

OH

O

CH 2

CH 2

CH 2

NH

C

NH 2

NH

lysine homoarginine arginine

Har