Dynamic docking and electron transfer between Zn-myoglobin and cytochrome b(5)

Area of Science:

• Biochemistry
• Biophysics
• Protein Interactions

Background:

• Electron transfer (ET) between proteins is vital for biological processes.
• Myoglobin (Mb) and cytochrome b5 (cyt b5) interactions are crucial for oxygen transport and metabolism.
• Understanding the factors influencing protein-protein interactions and ET is key to elucidating cellular mechanisms.

Purpose of the Study:

• To investigate the effect of neutralizing negative charges on Mb propionates on its interaction and ET with cyt b5.
• To explore how modifying Mb's heme environment impacts binding affinity and ET rates.
• To elucidate the mechanism of protein-protein interactions in electron transfer reactions.

Main Methods:

• Utilized zinc-substituted myoglobin (ZnMb) with modified heme propionates (diester and diamide) to neutralize charges.
• Measured photoinitiated ET rates over varying pH and ionic strengths.
• Employed isothermal titration calorimetry (ITC) and NMR spectroscopy to assess binding and conformational changes.
• Performed Brownian dynamics (BD) simulations and ET pathway calculations.

Main Results:

• Charge neutralization of Mb propionates increased the second-order ET rate constant with cyt b5 by up to several hundred-fold.
• Binding affinity between ZnMb and cyt b5 remained largely unchanged despite modifications.
• NMR studies showed minimal conformational changes in the Mb heme pocket.
• BD simulations supported a "dynamic docking" model where numerous weak interactions, rather than strong binding, facilitate ET.

Conclusions:

• The neutralization of Mb heme propionates enhances ET reactivity with cyt b5 without affecting binding affinity.
• Results support a "dynamic docking" paradigm, decoupling binding affinity from ET activity.
• This finding has significant physiological implications for redox processes involving Mb and hemoglobin (Hb) in vivo.