Computational structure prediction provides a plausible mechanism for electron transfer by the outer membrane protein Cyc2 from Acidithiobacillus ferrooxidans

Abstract

Cyc2 is the key protein in the outer membrane of Acidithiobacillus ferrooxidans that mediates electron transfer between extracellular inorganic iron and the intracellular central metabolism. This cytochrome c is specific for iron and interacts with periplasmic proteins to complete a reversible electron transport chain. A structure of Cyc2 has not yet been characterized experimentally. Here we describe a structural model of Cyc2, and associated proteins, to highlight a plausible mechanism for the ferrous iron electron transfer chain. A comparative modeling protocol specific for trans membrane beta barrel (TMBB) proteins in acidophilic conditions (pH ~ 2) was applied to the primary sequence of Cyc2. The proposed structure has three main regimes: Extracellular loops exposed to low-pH conditions, a TMBB, and an N-terminal cytochrome-like region within the periplasmic space. The Cyc2 model was further refined by identifying likely iron and heme docking sites. This represents the first computational model of Cyc2 that accounts for the membrane microenvironment and the acidity in the extracellular matrix. This approach can be used to model other TMBBs which can be critical for chemolithotrophic microbial growth.

Keywords: Rosetta; chemolithoautotrophy; heme protein; iron oxidation; transmembrane beta barrel.

FIGURE 1

Overview of metal oxidation by Acidithiobacillus ferrooxidans. (a). Overview of A. ferrooxidans metabolism when growing on ores containing sulfide minerals such as chalcopyrite (CuFeS₂). The bacterium oxidizes iron and reduced sulfur compounds, resulting in the solubilization of copper or other commercially valuable metal targets. Carbon dioxide is fixed via the Calvin cycle to 3‐phosphoglycerate (PGA). (b). Diagram of the electron transfer pathway used for extracellular iron oxidation. Electrons are transferred from the extracellular matrix, through the outer membrane (Cyc2), the periplasmic space (Rus and Cyc1) to coxABCD in the inner membrane. This is a simplified model of the downhill electron transfer pathway, as other periplasmic proteins may also be involved in this process. Under some conditions, electron transfer can be reversed, and iron reduction can also occur

FIGURE 2

Characterization of modeled Cyc2 protein and interactions with ligands: (a): Funnel plot for one hundred Cyc2 modeling trajectories using the modified set of membrane weights. The c‐alpha RMSD was measured in reference to the lowest‐scoring structure. (b): Lowest‐scoring Cyc2 protein model. Disks of spheres represent the phospholipid heads on the outer membrane where the top of the protein protrudes into the extracellular matrix and bottom of the protein (including the heme group) is in the periplasm. (c): Square planar iron chelating geometry for residues H119, D137, D138. (d): Tetrahedral iron binding geometry for Y262 and D308. E. Binding interaction between H16 and heme c

FIGURE 3

Predicted protein–protein interactions supporting the electron transport chain: (a): Docked models of the Cyc2‐rusticyanin‐Cyc1 pathway (where carbon backbone of Cyc2 is blue, rusticyanin in magenta, and Cyc1 is in yellow). Hemes, iron ions, and copper ions are indicated in sepia. (b): Predicted electron hopping pathway for electron transfer from the exterior of the cell into the periplasm of Acidithiobacillus ferrooxidans

FIGURE 4

Poisson–Boltzmann potential identifies possible iron binding pocket on the extracellular region of Cyc2: Poisson–Boltzmann potential for Cyc2, where blue indicates regions of positive potential (> +5 kT/e), whereas red depicts negative potential (<−5 kT/e). (a): labeled potential viewed from the side, where the upper disk represents phospholipid heads on the outer membrane facing the extracellular matrix and bottom disk represents the phospholipids facing the periplasm. There exists a region of high negative potential (circled in black) that may serve as a possible iron binding site. (b): Labeled potential as viewed from above, showing the protein structure protruding from the cell. Regions of high negative potential are circled in black. (c): Labeled potential as viewed from below the membrane, as would be observed from inside the cell. An annular space exists within the protein, inside which is predicted to have a high negative potential