Probing the ligand recognition and discrimination environment of the globin-coupled oxygen sensor protein YddV by FTIR and time-resolved step-scan FTIR spectroscopy

Abstract

YddV is a newly discovered signal transducer heme protein that recognizes O2 and CO. Structural differences in the ligand-bound heme complex in YddV reflect variations in catalytic regulation by O2 and CO. Time-resolved step-scan (TRS(2)) FTIR studies of the wild type and of the important in oxygen recognition and stability of the heme Fe(II)-O2 complex L65M, L65T, Y43A, Y43F and Y43W mutants were performed to determine the site-specific protein dynamics following carbon monoxide (CO) photodissociation. These mutations were designed to perturb the electrostatic field near the iron-bound gaseous ligand (CO) and also to allow us to investigate the communication pathway between the distal residues of the protein and heme. TRS(2)-FTIR spectra of YddV-heme-CO show that the heme propionates are in protonated and deprotonated states. Moreover, the rate of decay of the vibrations of amide I is on a time scale that coincides with the rate of rebinding of CO, which suggests that there is coupling between ligation dynamics in the distal heme environment and (i) relaxation of the protein backbone and (ii) the environment sensed by the heme propionates. The fast recombination rates in L65M, L65T and Y43W imply a significant role of L65 and Y43 in controlling the ligand dynamics. The implications of these results with respect to the role of the heme propionates and the charged or proton-donating residues in the distal pocket, which are crucial for stabilizing bound gaseous ligands, are discussed.

Ultrafast Spectroscopy Evidence for Picosecond Ligand Exchange at the Binding Site of a Heme Protein: Heme-Based Sensor YddV

“…This is different from WT YddV–heme, where NO dissociation does not lead to configurations favorable for water binding. This observation is in general agreement with the recently proposed role of Leu65 in controlling the CO binding dynamics to the heme …”

“…This observation is in general agreement with the recently proposed role of Leu65 in controlling the CO binding dynamics to the heme. 30 In conclusion, our results provide the first direct evidence of exchange between chemically different ligands at an intraprotein binding site occurring on the time scale of a few picoseconds. Here, the Fe(III)−NO bond is broken by photolysis, mimicking the native thermal dissociation process.…”

Protein Dynamics of the Sensor Protein HemAT as Probed by Time-Resolved Step-Scan FTIR Spectroscopy

“…2 A, trace A, the negative peaks at 1967 and 1925 cm À1 , which have been assigned to the neutral and strong H-bonded conformers of the heme Fe-CO complex, respectively, indicate that both conformers are photolabile. In addition to the CO modes, the amide I bands in the 1600-1700 cm À1 frequency range and that of the heme propionate at 1677 cm À1 are present (24,25). In Fig.…”

“…For the TRS 2 -FTIR measurements, 532 nm pulses from a Continuum Minilite II Nd:YAG laser (5 ns width, 10 Hz) were used as a pump light (4 mJ/pulse) to photolyze the HemAT-CO adducts. The TRS 2 -FTIR measurements were performed on a Bruker Vertex 80V spectrometer equipped with the step-scan option, as described previously (17,24). Briefly, a transistor-transistor-logic pulse provided by a digital-delay pulse generator (9524; Quantum Composers, Bozeman, MT) triggered in order the flashlamps, the Q-switch, and the FTIR spectrometer.…”

Probing the Role of the Heme Distal and Proximal Environment in Ligand Dynamics in the Signal Transducer Protein HemAT by Time-Resolved Step-Scan FTIR and Resonance Raman Spectroscopy

“…Time-resolved step-scan (TRS 2 ) FTIR spectroscopy has been established as a very potent technique that shows the distinction of different species with a fractional population in the percent range. − This is extremely useful when it comes to elucidating key processes for revealing information about the energy barriers governing internal migration and rebinding of the photodissociated ligand. Both A 0 and A 1 states in the CO-bound complex of HemAT are photolabile, ensuring that ligand binding reactions can be investigated by TRS 2 –FTIR spectroscopy.…”