Protein Interactions in Rhodopseudomonas palustris TIE-1 Reveal the Molecular Basis for Resilient Photoferrotrophic Iron Oxidation

Abstract

Rhodopseudomonas palustris is an alphaproteobacterium with impressive metabolic versatility, capable of oxidizing ferrous iron to fix carbon dioxide using light energy. Photoferrotrophic iron oxidation is one of the most ancient metabolisms, sustained by the pio operon coding for three proteins: PioB and PioA, which form an outer-membrane porin-cytochrome complex that oxidizes iron outside of the cell and transfers the electrons to the periplasmic high potential iron-sulfur protein (HIPIP) PioC, which delivers them to the light-harvesting reaction center (LH-RC). Previous studies have shown that PioA deletion is the most detrimental for iron oxidation, while, the deletion of PioC resulted in only a partial loss. The expression of another periplasmic HiPIP, designated Rpal_4085, is strongly upregulated in photoferrotrophic conditions, making it a strong candidate for a PioC substitute. However, it is unable to reduce the LH-RC. In this work we used NMR spectroscopy to map the interactions between PioC, PioA, and the LH-RC, identifying the key amino acid residues involved. We also observed that PioA directly reduces the LH-RC, and this is the most likely substitute upon PioC deletion. By contrast, Rpal_4085 demontrated significant electronic and structural differences from PioC. These differences likely explain its inability to reduce the LH-RC and highlight its distinct functional role. Overall, this work reveals the functional resilience of the pio operon pathway and further highlights the use of paramagnetic NMR for understanding key biological processes.

Keywords: HIPIP; Rhodopseudomonas; biological electron transfer; cytochrome c; paramagnetic NMR; photoferrotrophism; protein interactions.

Figure 1

Interactions between PioC and PioA. (A) Paramagnetically tailored ¹H¹⁵N-HSQC spectra of ¹⁵N PioC alone (blue) and with PioA (red). (B) Both chemical shift changes and line-broadening effects can be observed on HN resonances. (C) Protein map of changing resonances in the 3D structure of PioC. Electrostatic surface potential (−5 to +5 kT/e) of PioC (PDB ID: 6XYV) highlighting residues that interact with PioA. Electrostatic surface potentials were calculated using the APBS plugin in PyMOL [27,28].

Figure 2

Interactions between PioC and LH-RC. (A) Paramagnetically tailored 1H15N-HSQC spectra of 15N PioC alone (blue) and with LH-RC (red). (B) Both chemical shift changes and line-broadening effects can be observed on HN resonances. (C) Protein map of changing resonances in the 3D structure of PioC. Electrostatic surface potential (−5 to +5 kT/e) of PioC (PDB ID: 6XYV) highlighting residues that interact with LH-RC. Electrostatic surface potentials were calculated using the APBS plugin in PyMOL [27,28].

Figure 3

Paramagnetic NMR spectroscopy of PioC and Rpal_4085. (A) One-dimensional ¹H NMR spectra of reduced Rpal_4085 and temperature dependence of hyperfine shift of cysteine β-CH₂ and α-CH protons (A–F) outside the diamagnetic envelope. (B) One-dimensional ¹H NMR spectra of reduced PioC and temperature dependence of the hyperfine shift of cysteine β-CH₂ protons outside the diamagnetic envelope.

Figure 4

pH dependence of the reduction potentials of PioC and Rpal_4085. The lines were calculated for the coupled transfer of one proton with one electron considering pka_ox = 7.6 and pKa_red = 8.0 for PioC and pKa_ox = 5.5 and pKa_red = 8.2 for Rpal_4085 [35]. Of these pK_a values, only the pKa_ox of PioC was well-defined by the available data, whereas the others represent the upper bounds for the case of the pKa_ox of Rpal_4085 and the lower bounds for the pKa_red of both proteins that are compatible with the available experimental data. This allows for the calculation of the curves that illustrate the pH dependence of the potentials of the two proteins.

Figure 5

Structural and sequence comparison of PioC and Rpal_4085. (A) Structural alignment of PioC (green, PDB ID: 6XYV) and Rpal_4085 (magenta, AlphaFold model). (B) Sequence alignment performed with MUSCLE (EMBL-EBI) of PioC and Rpal_4085 where * (asterisk) indicates positions which have a single fully conserved amino-acid residue and : (colon) indicates conservation between groups of strongly similar properties. The PioA-interacting residues are highlighted in magenta, LH-RC interacting residues in green, and conserved residues in yellow. (C) Electrostatic surface potential (−5 to +5 kT/e) of PioC (PDB ID: 6XYV) and Rpal_4085 (AlphaFold model). Electrostatic surface potentials were calculated using the APBS plugin in PyMOL [32,37].

Figure 6

PioA transfers electrons directly to LH-RC. The observed UV-visible spectral changes reflect the oxidation of PioA in the presence of NADP⁺ and the RC upon sample illumination.