Background – What are cytochromes c, why are they important and how are they made?
Bacteria survive and thrive in nearly every niche on earth. They play important roles in our environment and can impact humans by keeping us healthy (i.e., the gut microbiome) or making us sick (i.e., pathogens). Bacteria’s resilience to survive in harsh environments comes partly from their ability to utilize many different energy sources, a process mediated by diverse electron transport chains1,2. A key component of many electron transport chains are cytochromes c. Cytochromes c are encoded by nearly all organisms from humans to bacteria. All cytochromes c have one or more covalently attached heme molecule(s) that are required for protein folding, function and confer unique redox properties to these proteins.
Figure 1. Schematic of cytochrome c biogenesis. The CXXCH motif is positioned in close proximity to heme. The cysteine thiols form a covalent thioether bond to the heme vinyl groups to form holocytochrome c, a process that is mediated by one of three cytochrome c biogenesis pathways called System I, II or III. AlphaFold 33 was used to model heme at the CXXCH motif of H. hepaticus cytochrome c oxidase (Cbb3) (UniProt ID: O87196).
Heme attachment or cytochrome c biogenesis (Fig. 1) occurs when heme is attached to a conserved motif (CXXCH) in cytochrome c. Cytochrome c biogenesis requires dedicated proteins that position heme along with the CXXCH motif to mediate heme attachment. Three pathways have been identified that can accomplish this process: System I (found mainly in bacteria, plant and protozoal mitochondria, and Archaea), System II (found mainly in Gram (+) bacteria and chloroplasts) and System III (found in eukaryotic mitochondria)4–7. These three pathways use different mechanisms to interact with, position and attach heme. Our lab is interested in understanding the similarities and differences between these fundamental biological pathways.
System II – What is it?
Here we focus on the System II bacterial cytochrome c biogenesis pathway, which is composed of two proteins CcsBA. Previous studies have proposed that CcsBA is a bi-functional enzyme that transports heme and attaches it to apocytochrome c8–11. CcsBA is essential in many organisms (i.e., they can’t live without it), so most studies on CcsBA are done by expressing the CcsBA protein of interest in E. coli. Because CcsBA is not required for E. coli to survive we can make mutations in CcsBA to understand how it works. Until this study, most biochemical and structural studies on CcsBA were performed with Helicobacter hepaticus CcsBA8–10. However, it has not been determined if all CcsBAs function the same way. This is an important question because CcsBAs from other bacteria have low sequence homology and variable sizes, therefore it not clear if what was previously learned from H. hepaticus CcsBA would be the same in other CcsBAs. However, stable and functional CcsBAs have been hard to recombinantly express in E. coli, limiting progress on these questions.
System II – How does it interact with heme?
In this paper we describe the successful recombinant expression of CcsBA from two bacterial pathogens, Helicobacter pylori and Campylobacter jejuni. H. pylori is a common cause of stomach ulcers12. C. jejuni is a leading cause of human diarrheal disease13. This technical advance allowed us to ask if the CcsBA-heme interaction is conserved across different bacteria. CcsBA must interact with heme to transport it across the bacterial membrane and then attach it to cytochrome c. We focused on heme binding in the WWD domain. This domain was shown to bind heme and be required for cytochrome c biogenesis in H. hepaticus CcsBA9,10. In our study, we used an approach called cysteine/heme crosslinking which allows us to ‘trap’ heme and study how it is bound by a protein9,14,15. Using this technique, we identified three amino acids in the WWD domains of H. pylori and C. jejuni CcsBA that interact with heme. This was very exciting because these same amino acids also interact with heme in the H. hepaticus CcsBA WWD domain (Fig. 2). This result shows that heme interacts with the WWD domain in a conserved manner in three different bacteria.
Figure 2. Heme binding is conserved in the WWD domain. A) AlphaFold 33 was used to model heme (red) into the predicted C. jejuni CcsBA structure (green, WWD domain in purple). Conserved residues that interact with heme are labeled. B-D) The conserved residues that interact with heme in the CcsBA WWD domains are indicated (residue 1-3). B) The AlphaFold 3 predicted structure of C. jejuni CcsBA WWD domain. C) The AlphaFold 3 predicted structure of H. pylori CcsBA WWD domain. D) The cryo-EM structure of H. hepaticus WWD domain (PDB 7S9Y10).
Why does heme binding in the WWD domain matter?
The CcsBA WWD domain binds heme in a conserved manner and is required for synthase function 8,9,16, meaning if the WWD domain is disrupted cytochromes c can not be made. In some bacteria, CcsBA is essential17,18 and bacteria can’t live without it. Therefore, we propose that the WWD domain could be a target for novel antibiotics. Importantly, current literature suggests that the way CcsBA attaches heme to cytochrome c is different than the way the humans make cytochrome c19–21. This supports the proposal that novel antibiotics targeting the CcsBA WWD domain will be specific to bacteria and won’t affect humans. Generally speaking, new therapeutics and new targets are critical to address increased resistance to current drug treatments. This is particularly important for H. pylori and C. jejuni that have been designated as high-priority pathogens for new antibiotics22,23. This study provides biochemical evidence for functional conservation in heme binding in the CcsBA WWD domain, suggesting that this domain could be a novel therapeutic target.
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