Dynamics of Lysine as a Heme Axial Ligand: NMR Analysis of the Chlamydomonas reinhardtii Hemoglobin THB1.
Biochemistry
; 56(4): 551-569, 2017 01 31.
Article
en En
| MEDLINE
| ID: mdl-28032976
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 (â¼3-4 × 103 s-1 at 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 by 15Nz-exchange spectroscopy are slow on the chemical shift time scale (101-102 s-1 at 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.
Texto completo:
1
Colección:
01-internacional
Banco de datos:
MEDLINE
Asunto principal:
Oxigenasas
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Hemoglobinas
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Chlamydomonas reinhardtii
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Proteínas Algáceas
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Hemo
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Lisina
Idioma:
En
Revista:
Biochemistry
Año:
2017
Tipo del documento:
Article
País de afiliación:
Estados Unidos