Engineering His(E7) affects the control of heme reactivity in Aplysia limacina myoglobin.
Aplysia limacina myoglobin lacks the distal histidine (His (E7)) and displays a ligand stabilization mechanism based on Arg(E10). The double mutant Val(E7)His-Arg(E10)Thr has been prepared to engineer the role of His(E7), typical of mammalian myoglobins, in a different globin framework. The 2.0 A crystal structure of Val(E7)His-Arg(E10)Thr met-Mb mutant reveals that the His(E7) side chain points out of the distal pocket, providing an explanation for the observed failure to stabilize the Fe(II) bound oxygen in the ferrous myoglobin. Moreover, spectroscopic analysis together with kinetic data on azide binding to met-myoglobin are reported and discussed in terms of the presence of a water molecule at coordination distance from the heme iron.
Federici
L
L
Department of Biochemical Sciences "A. Rossi Fanelli" and C.N.R. Center for Molecular Biology, University of Rome "La Sapienza", Piazzale Aldo Moro 5, Rome, 00185, Italy.
Savino
C
C
Musto
R
R
Travaglini-Allocatelli
C
C
Cutruzzolà
F
F
Brunori
M
M
D000818:Animals; D001048:Aplysia / Q000235:genetics / Q000378:metabolism*; D001386:Azides / Q000378:metabolism; D001665:Binding Sites / Q000235:genetics; D018360:Crystallography, X-Ray; D006418:Heme / Q000737:chemistry*; D006639:Histidine / Q000737:chemistry; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009211:Myoglobin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D011487:Protein Conformation; D015202:Protein Engineering; D055672:Static Electricity; D014907:Whales
D001386:Azides; D009211:Myoglobin; D006418:Heme; D006639:Histidine