The Unusual Heme Coordination of THB1, a Hemoglobin from Chlamydomonas Reinhardtii

Abstract

THB1 is a ∼15 kDa “truncated” hemoglobin (Hb) found in the cytoplasm of the unicellular green alga Chlamydomonas reinhardtii. As for other Hbs within photosynthetic microbes, THB1 is thought to be involved in the management of reactive oxygen/nitrogen species. To complement physiological studies aimed at determining the function of THB1, we are pursuing the characterization of the recombinant protein (rTHB1) [1] with special attention to the heme environment. At pH ∼ 5, ferric rTHB1 has an optical spectrum characteristic of a six-coordinate, high-spin complex with a histidine and a water molecule as axial ligands to the heme iron. When the pH is raised near neutrality, the protein undergoes a transition (apparent pKa of 6.5) to a six-coordinate, low-spin complex. Mutagenesis and NMR data collected in the diamagnetic ferrous state provide direct evidence for the ligation of a lysine (K53) on the distal side of the heme [1]. Here, we continue the study of this unusual iron coordination scheme. The distal lysine, like the distal histidine of cyanobacterial globins (GlbNs), facilitates electron transfer. However, unlike the histidine of GlbN, the distal lysine of THB1 does not protect ferric heme from oxidative damage caused by H2O2. Analysis of ferrous THB1 NMR data reveals a neutral lysine amino group with highly upfield-shifted 15N signal. Fast-exchange averaging of the two amino protons is a further indication of a dynamic distal coordination. Additional structural information was obtained from diffraction data collected at beamline X25 (National Synchrotron Light Source, Brookhaven National laboratory). Crystals of ferric rTHB1 grown at pH 9.5 diffracted to 1.9 A resolution (P6122 space group) and the data were used to inspect the structural factors allowing lysine coordination.[1] Johnson et al. (2014) Biochemistry 53:4573Supported by NSF grant MCB-1330488