P25: Effect of distal heme pocket mutations on neuroglobin nitrite reduction rates

Abstract

Neuroglobin is a highly conserved hemoprotein of uncertain physiological function that evolved from a common ancestor to hemoglobin and myoglobin. Unlike hemoglobin or myoglobin, which show a five-coordinate heme iron, neuroglobin shows a six-coordinate heme with proximal and distal histidines in the heme pocket directly bound to the heme iron. Previous work from our lab has shown that neuroglobin can reduce nitrite to nitric oxide. In order to assess the role in this reaction of the distal Histidine His64(E7) and other heme pocket residues-Phe28 (B10) and Val68 (E11), we studied the nitrite reductase activity of wild-type neuroglobin and mutations of the three aforementioned residues. The mutants H64Q, H64L, H64A, H64W, F28W, F28L, F28V, F28H, V68A, V68F and V68I were studied in terms of nitrite reductase activity, redox potential and autoxidation rates. All mutants except Val68Ala showed increases in nitrite reductase rates (1.5 to 25-fold increases for F28/V68 mutants; 60- to 10,000-fold increases for H64 mutants). Most His64 and Val68 mutations caused modest decreases in the autoxidation, except the V68A mutants which shows very fast autoxidation rates (half life of the oxy complex 26 s). The Phe28 mutations caused marked increases in the autoxidation rates (with half life of the oxy complexes below 1 min), indicating that only the Phe residue in the position B10 can stabilize the oxy-complex against autoxidation. His64 and Phe28 mutations caused a small decrease (10–20 mV) in the redox potential, with the exception of the H64Q mutation that increased the potential by about 45 mV. Val68 mutants showed slight increases. Nitrite reductase activity of neuroglobin appears to be more related to nitrite access to the heme than to other factors such as redox potential. It must be noted that the redox potentials of wt and mutant neuroglobins are in all cases significantly lower than those of hemoglobin or myoglobin. The similarities and differences between myoglobin and neuroglobin mutants will be discussed. Disclosure Supported by the Institute for Transfusion Medicine and the Hemophilia Center of Western Pennsylvania and grants from NIH (R01 HL096973-02 to M.G.) and the Competitive Medical Research Fund of the UPMC Health System (to J.T.).