The modular evolution of multiheme cytochromes c bucks the general trend observed in most proteins

Abstract

Multiheme cytochromes c are versatile enzymes which contribute to key steps in diverse biogeochemical cycles of chemical elements such as nitrogen, iron, and sulfur, and are also key players in microbial electrochemical technologies. Understanding how they evolved offers a blueprint on how nature tuned their sequence and heme c cofactors to accomplish this versatile chemistry. We previously focused our attention on the largest group of homologous multiheme cytochrome c and showed that these enzymes evolve by fusion and fission of cytochrome c modules. Here, we extended our analysis to all characterized multiheme cytochromes and found that, in general, the evolution of multiheme cytochromes is governed by the same principles of fusion and fission. In addition, here we show that fusion can also involve the incorporation of domains of other protein families and small peptide sequences that affect the heme coordination environment. We found that loss of heme‐binding motifs is more prevalent than gain. The addition of hemes to multiheme cytochromes c occurs exclusively by integrating heme‐binding peptides and not by gradual accumulation of mutations and is equally probable along the protein sequence. By contrast, heme loss occurs by the loss of heme‐binding peptides and the accumulation of point mutations. Notably, heme‐binding motif loss is disproportionately more prevalent at the position nearer to the N‐terminus. This observation contrasts with the general trend observed in proteins, which are usually more conserved at the N‐terminus, and likely reflects the way in which multiheme cytochromes c are assembled.