title: Dynamics of Lysine as a Heme Axial Ligand: NMR Analysis of the Chlamydomonas reinhardtii Hemoglobin THB1.
abstract: Nitrate metabolism in Chlamydomonas reinhardtii involves THB1, a monomeric hemoglobin thought to function as a nitric oxide dioxygenase (NOD). NOD activity requires dioxygen and nitric oxide binding followed by a one-electron oxidation of the heme iron and nitrate release. Unlike pentacoordinate flavohemoglobins, which are efficient NODs, THB1 uses two iron axial ligands: the conserved proximal histidine and a distal lysine (Lys53). As a ligand in both the oxidized (ferric) and reduced (ferrous) states, Lys53 is expected to lower the reorganization energy associated with electron transfer and therefore facilitate reduction of the ferric enzyme. In ferrous THB1, however, Lys53 must be displaced for substrate binding. To characterize Lys53 dynamics, THB1 was studied at various pH, temperatures, and pressures by NMR spectroscopy. Structural information indicates that the protein fold and Lys53 environment are independent of the oxidation state. High-pressure NMR experiments provided evidence that displacement of Lys53 occurs through fast equilibrium (∼3-4 × 103s-1at 1 bar, 298 K) with a low-population intermediate in which Lys53 is neutral and decoordinated. Once decoordinated, Lys53 is able to orient toward solvent and become protonated. The global lysine decoordination/reorientation/protonation processes measured by15Nz-exchange spectroscopy are slow on the chemical shift time scale (101-102s-1at pH ≈ 6.5, 298 K) in both iron redox states. Thus, reorientation/protonation steps in ferrous THB1 appear to present a significant barrier for dioxygen binding, and consequently, NOD turnover. The results illustrate the role of distal ligand dynamics in regulating the kinetics of multistep heme redox reactions.
authors.affiliation: T. C. Jenkins Department of Biophysics, Johns Hopkins University , Baltimore, Maryland 21218, United States.
authors.forename: Matthew R
authors.initials: MR
authors.lastname: Preimesberger
authors.affiliation: Biomolecular NMR Center, Johns Hopkins University , Baltimore, Maryland 21218, United States.
authors.forename: Ananya
authors.initials: A
authors.lastname: Majumdar
authors.affiliation: T. C. Jenkins Department of Biophysics, Johns Hopkins University , Baltimore, Maryland 21218, United States.
authors.forename: Juliette T J
authors.identifier: 0000-0003-1116-0053
authors.initials: JT
authors.lastname: Lecomte
mesh_terms: D020418:Algal Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D016825:Chlamydomonas reinhardtii / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D003001:Cloning, Molecular; D004579:Electron Transport; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D006418:Heme / Q000737:chemistry* / Q000378:metabolism; D006454:Hemoglobins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006639:Histidine / Q000737:chemistry / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008024:Ligands; D008239:Lysine / Q000737:chemistry* / Q000378:metabolism; D008958:Models, Molecular; D009569:Nitric Oxide / Q000737:chemistry / Q000378:metabolism; D019906:Nuclear Magnetic Resonance, Biomolecular; D010084:Oxidation-Reduction; D010100:Oxygen / Q000737:chemistry / Q000378:metabolism; D010105:Oxygenases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013696:Temperature; D013816:Thermodynamics
chemical_list: D020418:Algal Proteins; D006454:Hemoglobins; D008024:Ligands; D011994:Recombinant Proteins; D009569:Nitric Oxide; D006418:Heme; D006639:Histidine; D010105:Oxygenases; C114415:nitric oxide dioxygenase; D008239:Lysine; D010100:Oxygen