Iron release from recombinant N-lobe and mutants of human transferrin.
Mutations of kinetically active residues in the recombinant N-lobe of human transferrin may accelerate or retard release of iron from the protein to pyrophosphate, thereby providing means for exploring the individual roles of such residues in the concerted mechanisms of release. Using an established spectrofluorometric method and pyrophosphate as the required iron-sequestering agent, we have compared release from unaltered native transferrin and recombinant N-lobe half-transferrin to release from six N-lobe mutants, R124S, R124K, K206R, H207E, H249Y, and Y95H. Mutation of R124, which serves as a principal anchor for the synergistic carbonate anion ordinarily required for iron binding by transferrin, accelerates release. This effect is most marked at endosomal pH, 5.6, and is also evident at extracellular pH, 7.4, pointing to a critical and perhaps initiating role of carbonate in the release process. Mutation of K206 to arginine, or of H207 to glutamine, each lying in the interdomain cleft of the N-lobe, gives products mimicking the arrangements in lactoferrin. Release of iron from these two mutants, as from lactoferrin, is substantially slower than from unaltered recombinant N-lobe. Interdomain residues not directly involved in iron or anion binding may therefore participate in the control of iron release within the endosome. The H249Y mutant releases iron much more rapidly than its wild-type parent or any other mutant, possibly because of steric effects of the additional phenolic ring in the binding site. No simple explanation is available to account for a stabilizing effect of the Y95H mutation.(ABSTRACT TRUNCATED AT 250 WORDS)
Zak
O
O
Department of Physiology and Biophysics, Albert Einstein College of Medicine, Bronx, New York 10461, USA.
Aisen
P
P
Crawley
J B
JB
Joannou
C L
CL
Patel
K J
KJ
Rafiq
M
M
Evans
R W
RW
D006801:Humans; D007501:Iron / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014168:Transferrin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D011994:Recombinant Proteins; D014168:Transferrin; D007501:Iron