Eric A Johnson1, Miranda M Russo1, Dillon B Nye1, Jamie L Schlessman2, Juliette T J Lecomte3. 1. T. C. Jenkins Department of Biophysics, Johns Hopkins University, Baltimore, MD 21218, United States. 2. Chemistry Department, U.S. Naval Academy, Annapolis, MD 21402, United States. 3. T. C. Jenkins Department of Biophysics, Johns Hopkins University, Baltimore, MD 21218, United States. Electronic address: lecomte_jtj@jhu.edu.
Abstract
BACKGROUND: The nuclear genome of Chlamydomonas reinhardtii encodes a dozen hemoglobins of the truncated lineage. Four of these, named THB1-4, contain a single ~130-residue globin unit. THB1, which is cytoplasmic and capable of nitric oxide dioxygenation activity, uses a histidine and a lysine as axial ligands to the heme iron. In the present report, we compared THB2, THB3, and THB4 to THB1 to gain structural and functional insights into algal globins. METHODS: We inspected properties of the globin domains prepared by recombinant means through site-directed mutagenesis, electronic absorption, CD, and NMR spectroscopies, and X-ray crystallography. RESULTS: Recombinant THB3, which lacks the proximal histidine but has a distal histidine, binds heme weakly. NMR data demonstrate that the recombinant domains of THB2 and THB4 coordinate the ferrous heme iron with the proximal histidine and a lysine from the distal helix. An X-ray structure of ferric THB4 confirms lysine coordination. THB1, THB2, and THB4 have reduction potentials between -65 and -100 mV, are capable of nitric oxide dioxygenation, are reduced at different rates by the diaphorase domain of C. reinhardtii nitrate reductase, and show different response to peroxide treatment. CONCLUSIONS: Three single-domain C. reinhardtii hemoglobins use lysine as a distal heme ligand in both Fe(III) and Fe(II) oxidation states. This common feature is likely related to enzymatic activity in the management of reactive oxygen species. GENERAL SIGNIFICANCE: Primary structure analysis of hemoglobins has limited power in the prediction of heme ligation. Experimental determination reveals variations in this essential property across the superfamily.
BACKGROUND: The nuclear genome of Chlamydomonas reinhardtii encodes a dozen hemoglobins of the truncated lineage. Four of these, named THB1-4, contain a single ~130-residue globin unit. THB1, which is cytoplasmic and capable of nitric oxide dioxygenation activity, uses a histidine and a lysine as axial ligands to the heme iron. In the present report, we compared THB2, THB3, and THB4 to THB1 to gain structural and functional insights into algal globins. METHODS: We inspected properties of the globin domains prepared by recombinant means through site-directed mutagenesis, electronic absorption, CD, and NMR spectroscopies, and X-ray crystallography. RESULTS: Recombinant THB3, which lacks the proximal histidine but has a distal histidine, binds heme weakly. NMR data demonstrate that the recombinant domains of THB2 and THB4 coordinate the ferrous hemeiron with the proximal histidine and a lysine from the distal helix. An X-ray structure of ferricTHB4 confirms lysine coordination. THB1, THB2, and THB4 have reduction potentials between -65 and -100 mV, are capable of nitric oxide dioxygenation, are reduced at different rates by the diaphorase domain of C. reinhardtiinitrate reductase, and show different response to peroxide treatment. CONCLUSIONS: Three single-domain C. reinhardtii hemoglobins use lysine as a distal heme ligand in both Fe(III) and Fe(II) oxidation states. This common feature is likely related to enzymatic activity in the management of reactive oxygen species. GENERAL SIGNIFICANCE: Primary structure analysis of hemoglobins has limited power in the prediction of heme ligation. Experimental determination reveals variations in this essential property across the superfamily.
Authors: M Couture; T K Das; H C Lee; J Peisach; D L Rousseau; B A Wittenberg; J B Wittenberg; M Guertin Journal: J Biol Chem Date: 1999-03-12 Impact factor: 5.157
Authors: Eric A Johnson; Selena L Rice; Matthew R Preimesberger; Dillon B Nye; Lukas Gilevicius; Belinda B Wenke; Jason M Brown; George B Witman; Juliette T J Lecomte Journal: Biochemistry Date: 2014-07-09 Impact factor: 3.162
Authors: Igor Efimov; Gary Parkin; Elizabeth S Millett; Jennifer Glenday; Cheuk K Chan; Holly Weedon; Harpreet Randhawa; Jaswir Basran; Emma L Raven Journal: FEBS Lett Date: 2014-01-17 Impact factor: 4.124