3‐Fluorotyrosine as a Complementary Probe of Hemoglobin Structure and Dynamics: A ¹⁹F‐NMR Study of Synechococcus sp. PCC 7002 GlbN

Abstract

The hemoglobin from the cyanobacterium Synechococcus sp. PCC 7002 (GlbN) contains three tyrosines (Tyr5, Tyr22, and Tyr53), each of which undergoes a structural rearrangement when the protein binds an exogenous ligand such as cyanide. We explored the use of 3-fluorotyrosine and (19)F-NMR spectroscopy for the characterization of GlbN. Assignment of (19)F resonances in fluorinated GlbN (GlbN*) was achieved with individual Tyr5Phe and Tyr53Phe replacements. We observed marked variations in chemical shift and linewidth reflecting the dependence of structural and dynamic properties on oxidation state, ligation state, and covalent attachment of the heme group. The isoelectronic complexes of ferric GlbN* with cyanide and ferrous GlbN* with carbon monoxide gave contrasting spectra, the latter exhibiting heterogeneity and enhanced internal motions on a microsecond-to-millisecond time scale. The strength of the H-bond network involving Tyr22 (B10) and bound cyanide was tested at high pH. 3-Fluorotyrosine at position 22 had a pK(a) value at least 3 units higher than its intrinsic value, 8.5. In addition, evidence was found for long-range communication among the tyrosine sites. These observations demonstrated the utility of the 3-fluorotyrosine approach to gain insight in hemoglobin properties.

Influence of Heme Post-Translational Modification and Distal Ligation on the Backbone Dynamics of a Monomeric Hemoglobin

“…Because of the stability of the complexes they form, they are also studied as surrogates for O 2 -bound states, although there are obvious limitations to this approach. − In GlbN-X, the similarities between the cyanomet and carbonmonoxy complexes included tertiary structure and picosecond to nanosecond time scale motions but broke down when microsecond to millisecond time scale motions were considered, presumably because of weak interactions between the protein and carbon monoxide. Distinct cyanomet and carbonmonoxy GlbN properties were also evidenced by 19 F NMR spectroscopy in protein containing 3-fluorotyrosine at position B10, the cyanomet state yielding sharp lines and the carbonmonoxy state yielding a broad and heterogeneous spectrum. These side chain observations reinforce a connection between the microsecond to millisecond time scale dynamics of GlbN and the nature of the distal ligand.…”

Fluorescence Modulation of Green Fluorescent Protein Using Fluorinated Unnatural Amino Acids

“…Fluorotyrosine UAAs are of particular interest in the tuning of GFP fluorescence because they are isosterically similar to the natural tyrosine residue, but have dramatically different electronic properties ( Table 1 ). Previously, 3-fluorotyrosine ( 1 ) has been employed to determine the structure and kinetics of proteins such as hemoglobin [ 17 ], organophosphate hydrolase [ 18 ], and human manganese superoxide dismutase [ 19 ]. Moreover, its ability to promote and propagate radicals has been explored in the study of numerous reactive tyrosine radical mechanisms [ 20 ].…”

The 2/2 hemoglobin from the cyanobacterium Synechococcus sp. PCC 7002 with covalently attached heme: Comparison of X‐ray and NMR structures

“…Among the properties important to a catalytic role are predisposition for heme PTM, structural plasticity, and ability to transfer electrons . To interpret our in vitro results, we have solved the structure of heme‐modified GlbN in the ferric, bis ‐histidine state by NMR methods (PDB ID: 2KSC) and obtained partial solution structure information for the cyanide‐bound state .…”