1：Experimental Design and Method Selection:

The study involved preparing cytochrome b562 co-assemblies and heterodimers with Fe and Zn porphyrins using metalloporphyrin–heme pocket interactions and hydrogen-bond networks. Laser flash photolysis was used to monitor photoinduced electron transfer kinetics, and Marcus theory was applied for analysis.

2：Sample Selection and Data Sources:

Cytochrome b562 and its mutants (N80C, R98A/N80C, D73A/N80C) were expressed in E. coli and purified. Apo-proteins were prepared, and co-assemblies/heterodimers were formed by mixing FeP and ZnP-containing assemblies or via maleimide-thiol conjugation.

3：List of Experimental Equipment and Materials:

Equipment included ESI-TOF MS (Bruker Daltonics micrOTOF II), UV-vis spectrometers (Shimadzu UV-3150, UV-2550, JASCO V-670), CD spectrometer (JASCO J-820S), fluorescence spectrometer (JASCO FP-8600), SEC system (?KTApurifier, GE Healthcare), pH meter (Horiba F-52), glove box (MBraun UNILab), laser flash photolysis setup with Nd:YAG laser, Xenon lamp, monochromator, photomultiplier (Unisoku), oscilloscope (Tektronix TDS3012), and software (KaleidaGraph). Materials included Fe and Zn protoporphyrin IX complexes, Cyt b562 mutants, buffers, and chemicals like DTT and potassium ferricyanide.

4：Experimental Procedures and Operational Workflow:

Co-assemblies were prepared by mixing FeP and ZnP assemblies and equilibrating; heterodimers were formed via conjugation and purified by gel filtration. Samples were degassed and placed in quartz cells for laser flash photolysis at 532 nm, monitoring transient absorptions at specific wavelengths (e.g., 460 nm for triplet states, 680 nm for ZnP radical cation).

5：Data Analysis Methods:

Transient absorption decays were fitted using exponential equations in KaleidaGraph to extract kinetic constants (kCS, kCR, ki). Marcus equation was used to analyze ET kinetics, considering driving force, reorganization energy, and electronic coupling.