Structural and functional characterization of sperm whale myoglobin mutants: role of arginine (E10) in ligand stabilization.
1H NMR and ligand-binding data were used to assess the role of residue Arg(E10) in ligand stabilization of several site-directed mutants, all carrying the His(E7) to Val substitution, obtained using a synthetic sperm whale myoglobin gene. Arg(E10) was previously found to form a hydrogen bond with the ligand in fluoro-, azido- and cyanomet derivatives of Aplysia limacina myoglobin, which lacks the distal His(E7) [Qin, J., La Mar, G. N., Ascoli, F., Bolognesi, M., & Brunori, M. (1992) J. Mol. Biol. 224, 891-897]. NMR analysis of the paramagnetically induced relaxation, hyperfine shift patterns, and dipolar connectivities shows that Arg(E10) also falls into the distal pocket in the engineered sperm whale myoglobin mutants and resides at an H-bonding distance from the Fe(3+)-bound cyanide. The rate constant for cyanide dissociation from the ferrous derivative was determined by stopped-flow experiments; the ligand stabilization achieved by Arg(E10) is similar to that exerted by His(E7) in wild-type sperm whale myoglobin, and both are very different from the His(E7)Val single mutant. Contrary to that for the wild-type, the cyanide dissociation rate constant for the mutant containing Arg(E10) is essentially independent of pH (from 6 to 9), as expected on the basis of the guanidinium group of Arg having a pK > 10. This finding is consistent with the NMR data in which the chemical shift of the Arg(E10) N epsilon H is insensitive to pH (6-9), as is also observed in Aphysia limacina cyanometmyoglobin.(ABSTRACT TRUNCATED AT 250 WORDS)
Allocatelli
C T
CT
Department of Biochemical Sciences A. Rossi Fanelli, University of Rome La Sapienza, Italy.
Cutruzzolà
F
F
Brancaccio
A
A
Brunori
M
M
Qin
J
J
La Mar
G N
GN
D000595:Amino Acid Sequence; D000818:Animals; D001120:Arginine / Q000502:physiology; D003486:Cyanides; D008024:Ligands; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009211:Myoglobin / Q000737:chemistry* / Q000235:genetics / Q000502:physiology; D011485:Protein Binding; D017433:Protein Structure, Secondary; D013329:Structure-Activity Relationship; D014867:Water; D014907:Whales
D003486:Cyanides; D008024:Ligands; D009211:Myoglobin; D014867:Water; D001120:Arginine