MUTAGENESIS OF THE CONSERVED LYSINE-14 OF CYTOCHROME-C-550 FROM THIOBACILLUS-VERSUTUS AFFECTS THE PROTEIN-STRUCTURE AND THE ELECTRON SELF-EXCHANGE RATE
M. Ubbink et Gw. Canters, MUTAGENESIS OF THE CONSERVED LYSINE-14 OF CYTOCHROME-C-550 FROM THIOBACILLUS-VERSUTUS AFFECTS THE PROTEIN-STRUCTURE AND THE ELECTRON SELF-EXCHANGE RATE, Biochemistry, 32(50), 1993, pp. 13893-13901
The lysine residue K14 of cytochrome c-550 of Thiobacillus versutus ha
s been mutated to a glutamine (Q) and a glutamate (E) residue. These m
utations have a minimal effect on the pK(a) for replacement of the met
hionine ligand (the ''alkaline transition''), indicating that a presum
ptive salt bridge between K14 and E11 does not help stabilize the nati
ve form. This is in contrast with mitochondrial cytochrome c, where th
e homologous K13 forms a structurally important salt bridge with gluta
mate 90. The NMR signals of protons close to the heme iron in wild-typ
e and mutant ferricytochrome c-550 shift considerably with increasing
ionic strength. These effects resemble those seen in mitochondrial cyt
ochrome c upon addition of salt and upon complex formation with redox
partners. It is likely that electrostatic screening of positive charge
s near the heme crevice leads to a slight redistribution of the electr
on density in the heme. At low ionic strength the NMR spectrum of wild
-type cytochrome c-550 shows broad peaks. Line widths decrease upon ad
dition of salt up to 200 mM. In K14Q and K14E cytochrome c-550 the lin
e widths are much smaller at low ionic strength. Wild-type cytochrome
c-550 may exist in two exchanging conformations, one o which may repre
sent a more open (non-native) form, in analogy with cytochrome c. Howe
ver, in the case of cytochrome c-550 this non-native form does not sho
w ligand replacement. The electron self-exchange rates of wild type an
d mutants have been determined as a function of the ionic strength. At
zero ionic strength these rates are 2(1) X 10(2) M-1 s-1 (wild type),
7(2) x 10(3) M-1 s-1 (K14Q), and 1.2(0.4) x 10(4) M-1 s-1 (K14E); wit
h increasing ionic strength the differences between these rates decrea
se. In the presence of 0.55 M NaCl the rate for wild-type cytochrome c
-550 is 1.0(0.3) x 10(5) M-1 s-1.