Alkyl isocyanides serve as transition state analogues for ligand entry and exit in myoglobin.

Alkyl isocyanides (CNRs) identify pathways for diatomic ligand movement into and out of Mb, with their side chains acting as transition state analogues. The bound alkyl groups point either into the back of the distal pocket (in conformation, nu(CN) approximately 2070-2090 cm(-1)), which allows hydrogen bond donation from His64(E7) to the isocyano group, or toward solvent through an open His(E7) channel (out conformation, nu(CN) approximately 2110-2130 cm(-1)), which prevents polar interactions with the isocyano atoms. Fractions of the in conformer (F(in)) were measured by FTIR spectroscopy for methyl through n-pentyl isocyanide bound to a series of 20 different distal pocket mutants of sperm whale myoglobin and found to be governed by the ease of rotation of the His(E7) side chain, distal pocket volume and steric interactions, and, for the longer isocyanides, the unfavorable hydrophobic effect of placing their terminal carbon atoms into the solvent phase in the out conformation.

Distal histidine stabilizes bound O2 and acts as a gate for ligand entry in both subunits of adult human hemoglobin.

The role of the distal histidine in regulating ligand binding to adult human hemoglobin (HbA) was re-examined systematically by preparing His(E7) to Gly, Ala, Leu, Gln, Phe, and Trp mutants of both Hb subunits. Rate constants for O(2), CO, and NO binding were measured using rapid mixing and laser photolysis experiments designed to minimize autoxidation of the unstable apolar E7 mutants. Replacing His(E7) with Gly, Ala, Leu, or Phe causes 20-500-fold increases in the rates of O(2) dissociation from either Hb subunit, demonstrating unambiguously that the native His(E7) imidazole side chain forms a strong hydrogen bond with bound O(2) in both the alpha and beta chains (DeltaG(His(E7)H-bond) approximately -8 kJ/mol).

NIX is required for programmed mitochondrial clearance during reticulocyte maturation.

The regulated clearance of mitochondria is a well recognized but poorly understood aspect of cellular homeostasis, and defects in this process have been linked to aging, degenerative diseases, and cancer. Mitochondria are recycled through an autophagy-related process, and reticulocytes, which completely eliminate their mitochondria during maturation, provide a physiological model to study this phenomenon. Here, we show that mitochondrial clearance in reticulocytes requires the BCL2-related protein NIX (BNIP3L).

Role of erythropoietin receptor signaling in Friend virus-induced erythroblastosis and polycythemia.

Friend virus is an acutely oncogenic retrovirus that causes erythroblastosis and polycythemia in mice. Previous studies suggested that the Friend virus oncoprotein, gp55, constitutively activates the erythropoietin receptor (EPOR), causing uncontrolled erythroid proliferation. Those studies showed that gp55 confers growth factor independence on an interleukin-3 (IL-3)-dependent cell line (Ba/F3) when the EPOR is coexpressed.