Increasing the conformational stability by replacement of heme axial ligand in c-type cytochrome.
To investigate the role of the heme axial ligand in the conformational stability of c-type cytochrome, we constructed M58C and M58H mutants of the red alga Porphyra yezoensis cytochrome c(6) in which the sixth heme iron ligand (Met58) was replaced with Cys and His residues, respectively. The Gibbs free energy change for unfolding of the M58H mutant in water (DeltaG degrees (unf)=1.48 kcal/mol) was lower than that of the wild-type (2.43 kcal/mol), possibly due to the steric effects of the mutation on the apoprotein structure. On the other hand, the M58C mutant exhibited a DeltaG degrees (unf) of 5.45 kcal/mol, a significant increase by 3.02 kcal/mol compared with that of wild-type. This increase was possibly responsible for the sixth heme axial bond of M58C mutant being more stable than that of wild-type according to the heme-bound denaturation curve. Based on these observations, we propose that the sixth heme axial ligand is an important key to determine the conformational stability of c-type cytochromes, and the sixth Cys heme ligand will give stabilizing effects.
Satoh
Tadashi
T
Department of Biological Chemistry, College of Bioresource Sciences, Nihon University, Kameino 1866, Fujisawa, 252-8510, Kanagawa, Japan.
Itoga
Akito
A
Isogai
Yasuhiro
Y
Kurihara
Masaaki
M
Yamada
Seiji
S
Natori
Miwa
M
Suzuki
Noriko
N
Suruga
Kohei
K
Kawachi
Ryu
R
Arahira
Masaomi
M
Nishio
Toshiyuki
T
Fukazawa
Chikafusa
C
Oku
Tadatake
T
D001483:Base Sequence; D003580:Cytochromes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D045348:Cytochromes f; D017931:DNA Primers; D006418:Heme / Q000737:chemistry / Q000378:metabolism*; D008024:Ligands; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D011489:Protein Denaturation; D000461:Rhodophyta / Q000201:enzymology; D013056:Spectrophotometry, Ultraviolet
D003580:Cytochromes; D017931:DNA Primers; D008024:Ligands; D006418:Heme; D045348:Cytochromes f