Dual Axial-Ligand Substitution of the P-Proximal Heme Impairs Donor-Side Electron Transfer in Rubrivivax gelatinosus

The reaction center (RC)-bound tetraheme cytochrome of Rubrivivax gelatinosus provides a rapid and buffered electron-delivery pathway to the photooxidized primary donor P⁺. We examined a pufC M238V/H253A double mutant that replaces both native axial ligands of the P-proximal heme. Donor-side function was characterized in intact cells by time-resolved P/P⁺ absorption changes, cytochrome photooxidation, delayed fluorescence, and near-infrared spectroscopy. During rectangular excitation, P⁺ accumulated much faster in the mutant than in wild type (half-rise time ≈130 μs versus ≈2 ms), whereas terbutryn-restricted single-turnover measurements indicated that rapid initial P⁺ reduction was retained. Terbutryn-normalized cytochrome oxidation reached approximately four turnover equivalents in wild type but only about two in the mutant, consistent with reduced donor-side redox capacity. The mutant also showed increased delayed-fluorescence amplitude, a marked increase in the spectral contribution of LH2 relative to the LH1–RC core and a two- to threefold increase in the growth doubling time. A resolved kinetic model indicates that impaired replenishment of the P-proximal heme from the upstream donor chain can account qualitatively for the accelerated P⁺ accumulation. Thus, the double axial-ligand substitution weakens donor-side redox buffering without abolishing the rapid initial electron-donation step and is associated with broader remodeling of the photosynthetic apparatus.