Regulation of cytoglobin-lipid interactions by surface mutations

Cytoglobin is a heme protein of the globin family. Despite sequence homology with hemoglobin and myoglobin, cytoglobin shows a different heme coordination pattern. Instead of the single histidine, pentacoordinated heme iron of hemoglobin and myoglobin, cytoglobin shows bis‐histidine, hexacoordinate heme iron ligation. This is similar to the behavior observed in the related protein neuroglobin. Although conserved in most vertebrates, the function of cytoglobin is yet unknown. The changes in heme coordination lead to properties not consistent with an oxygen transport role, and a number of functions such as scavenging of reactive oxygen species, nitrite reduction, and nitric oxide dioxygenation have been proposed. In previous work, we have shown that cytoglobin has an intrinsic peroxidase activity that can be modulated by lipids and by the oxidation of two surface cysteines, suggesting a role of cytoglobin regulated by oxidative stress. We have proposed a model for lipid binding based on computational studies, highlighting several cytoglobin residues that can modulate the protein‐lipid interactions. Here we studied the lipid binding properties of several cytoglobin mutations selected from our previous models. We determined the lipid binding properties of the wild‐type cytoglobin and mutants by difference spectroscopy. The following mutations were studied: T91D, R84E, K116E, L108Q, and V105Y. In order to test whether the mutations cause significant changes in heme reactivity, we determined the autoxidation rates of the cytoglobin ferrous‐oxy species and the rate of nitrite reduction by the cytoglobin deoxy species. These reactions were monitored by the spectral changes due to the decrease in ferrous oxy species and the build‐up of the ferric form of the protein (autoxidation) or the transition from the deoxy species to the ferrous‐nitrosyl complex (nitrite reduction). Previous studies on neuroglobin indicate that the autoxidation rates correlate with the heme redox potential whereas the nitrite reduction rates are sensitive to changes in heme pocket ligand accessibility. The study of the nitrite reduction rates and autoxidation rates indicates that all mutants are show values within 50% of the wild type values, except for a 3‐fold increase in nitrite reduction rate for the V105Y mutant. All the mutations caused significant increases in the Kd for oleic acid as compared to the wild‐type enzyme (Kd=0.8 μM). However, the magnitude allowed classification of the mutants in three groups‐those showing minor changes in lipid affinity (V105Y, 4.8 μM; T91D, 5.7 μM), moderate changes (L108Q, 14 μM) or large changes (K116E, 62 μM; R84E, 170 μM). These results suggest that interaction of the positively charged R84 and K116 side chains with the polar carboxylic group of the lipid greatly contributes to the lipid binding interaction, whereas the hydrophobic portion of the lipid interacts with other regions of the protein probably included the L108 side chain. In summary, we show that surface mutation K116E and R84E can disrupt lipid binding to cytoglobin without altering heme reactivity. Our results support the use of these mutations in cell or animal models to study the effect of cytoglobin‐mediated peroxidation without impacting other possible cytoglobin roles, thus serving as new tools for the study of cytoglobin function.