Using PROPKA with a pre-charged heme cofactor at a protein–protein interface

[omardebei]

Hello everyone,

I would like to ask for advice on a workflow involving PDB2PQR/PROPKA applied to a heme-containing protein–protein complex.

System description:

• The system is a protein–protein complex with a heme (HEM) cofactor located at the interface.
• I can provide the heme as a MOL2 file in which:
  • all hydrogen atoms are already present,
  • atomic charges have been assigned externally (e.g. AmberTools/OpenBabel),
  • geometry and protonation state are fixed and chemically reasonable.

Goal:
My goal is to use PDB2PQR primarily to:

1. Assign protonation states to protein residues using PROPKA,
2. Generate a PQR file for electrostatic calculations (e.g. APBS),

while treating the heme as a fixed cofactor that:

• contributes to the electrostatic environment,
• but should not be modified (no charge reassignment or protonation changes).

Is there a recommended or supported way to:

• include a pre-charged heme MOL2 in PDB2PQR
• while still allowing PROPKA to titrate the surrounding protein residues?

Or is the intended/best-practice workflow to:

• exclude the heme during the PROPKA step,
• and merge it back into the final PQR prior to electrostatic calculations?

If anyone has experience with similar metallo-cofactors (heme, metal centers, prosthetic groups) or has developed a practical workaround, I would greatly appreciate your insights.

Thanks in advance for your help!