Evolutionary Relationships Between Low Potential Ferredoxin and Flavodoxin Electron Carriers

Abstract

Proteins from the ferredoxin (Fd) and flavodoxin (Fld) families function as low potential electrical transfer hubs in cells, at times mediating electron transfer between overlapping sets of oxidoreductases. To better understand protein electron carrier (PEC) use across the domains of life, we evaluated the distribution of genes encoding [4Fe-4S] Fd, [2Fe-2S] Fd, and Fld electron carriers in over 7,000 organisms. Our analysis targeted genes encoding small PEC genes encoding proteins having ≤200 residues. We find that the average number of small PEC genes per Archaea (~13), Bacteria (~8), and Eukarya (~3) genome varies, with some organisms containing as many as 54 total PEC genes. Organisms fall into three groups, including those lacking genes encoding low potential PECs (3%), specialists with a single PEC gene type (20%), and generalists that utilize multiple PEC types (77%). Mapping PEC gene usage onto an evolutionary tree highlights the prevalence of [4Fe-4S] Fds in ancient organisms that are deeply rooted, the expansion of [2Fe-2S] Fds with the advent of photosynthesis and a concomitant decrease in [4Fe-4S] Fds, and the expansion of Flds in organisms that inhabit low-iron host environments. Surprisingly, [4Fe-4S] Fds present a similar abundance in aerobes as [2Fe-2S] Fds. This bioinformatic study highlights understudied PECs whose structure, stability, and partner specificity should be further characterized.

Keywords: electron transfer; evolution; ferredoxin; flavin mononucleotide; flavodoxin; iron-sulfur cluster; oxidative stress; oxidoreductase.

FIGURE 1 |

Average PEC gene counts across the domains of life. The average number of PEC genes containing motifs that are characteristic of [4Fe-4S] Fds (red), [2Fe-2S] Fds (green), and Flds (blue) in each domain. The number of genomes analyzed within each domain is shown on top of each bar.

FIGURE 2 |

Numbers of genomes having different abundances of PEC genes. Number of organisms with different (A) [4Fe-4S] Fd (red), (B) [2Fe-2S] Fd (green), and (C) Fld (blue) gene counts. (D) Number of organisms with different total numbers of PEC genes (black). The data shown was obtained by summing up the Fld, [2Fe-2S] Fd, and [4Fe-4S] Fd counts in each genome.

FIGURE 3 |

Pairwise abundance of PEC genes within each genome. The abundances of (A) [2Fe-2S] Fd and Fld, (B) [4Fe-4S] Fd and Fld, and (C) [4Fe-4S] Fd and [2Fe-2S] Fd genes are plotted as heat maps. The density of organisms having each pairwise count is shown using a viridis color gradient.

FIGURE 4 |

Relative abundances of PEC specialist and generalists. The relative percentage of PEC gene counts for all three families are plotted with the density of organisms at each coordinate illustrated by a viridis color gradient. White hexagons represent combinations that were not observed.

FIGURE 5 |

PEC gene abundance mapped onto an evolutionary tree. Bars are used to illustrate the total PEC gene counts of the three different PEC families at the leaves in a stacked bar graph, including [4Fe-4S] Fds (red), [2Fe-2S] Fds (green), and Flds (blue). The domain for each organism is depicted by shading internal to the stacked bars: Eukaryotes (Light gray), Archaea (dark gray), and Bacteria (black). The major phyla and classes of organisms represented in the tree are visualized internal to the domains using the colored bars as noted in the key. Phyla with only one representative are labeled with white bars.

FIGURE 6 |

Relationship between environmental niche and PEC use. The average PEC gene counts for organisms having different (A) O₂ requirements and (B) growth temperatures. Colors represent the counts of [4Fe-4S] Fd (red), [2Fe-2S] Fd (green), and Fld (blue) gene abundances. The number of genomes of each type is shown adjacent to the bars.

FIGURE 7 |

PEC counts sorted by temperature and oxygen niche. The average numbers of (A) [4Fe-4S] Fd (B), [2Fe-2S] Fd, and (C) Fld gene counts per genome are plotted as a function of O₂ requirement and optimal growth temperature. (D) Genome counts from each environmental niche are plotted as a function of O₂ requirement and optimal growth temperature. O₂ requirements shown on the x axis are abbreviated as obligate anaerobe (OAn), anaerobe (An), facultative (F), microaerophilic (M), aerobic (A), and obligate aerobic (OA). Growth temperatures shown on the y axis are abbreviated as hyperthermophile (H), thermophile (T), mesophile (M), and psychrophile (P). N/A denotes not applicable when organisms were not observed.

FIGURE 8 |

Relationship between environmental pH and PEC gene counts. The distribution of PEC gene counts is normalized by the number of organisms found to grow at each pH value to obtain weighted averages of each PEC type. Analysis of the means revealed no significant pairwise differences using a paired two-tail t-test (p = 0.126 for [2Fe-2S] Fd and Fld comparison, p = 0.163 for [4Fe-4S] Fd and Fld comparison, and p = 0.75 for [2Fe-2S] and [4Fe-4S] Fd comparison).

FIGURE 9 |

Relationship between PEC length and number of PECs per organism. The abundances of (A) [4Fe-4S] Fds, (C) [2Fe-2S] Fds, and (E) Flds of different lengths encoded by genomes having different total numbers of PEC genes. Violin plots show the relative abundance of PECs having different sizes in each bin. The extrema are marked by horizontal edge lines at the ends of the vertical bars, and the average length is marked by an internal horizontal line. The number of genes within each violin plot is visualized using a viridis color gradient. For comparison, the lengths of (B) [4Fe-4S] Fd, (D) [2Fe-2S] Fd, and (F) Fld structures deposited in the Protein Data Bank (Berman, 2000). The number of structures used to generate these plots are noted at the bottom of each panel.

FIGURE 10 |

PEC length distributions in organisms having different O₂ requirements. The lengths of (A) [4Fe-4S] Fd, (B) [2Fe-2S] Fd, and (C) Fld genes in organisms having distinct O₂ requirements. Growth requirements are abbreviated as obligate anaerobe (OAn), anaerobe (An), facultative (F), microaerophilic (M), aerobic (A), and obligate aerobic (OA). The extrema are marked by the horizontal bars at the edges of the vertical bars, and the average length is noted with an internal horizontal bar. The number of individual genes in each category is listed below each plot.

FIGURE 11 |

PEC length distributions in organisms having different growth temperatures. The lengths of (A) [4Fe-4S] Fds, (B) [2Fe-2S] Fds, and (C) Flds found in organisms having different optimal growth temperatures is shown using violin plots. Optimal growth temperatures are abbreviated as hyperthermophile (H), thermophile (T), mesophile (M), and psychrophile (P). The extrema are noted with horizontal bars at the edges, and the average is noted with an internal horizontal bar. The number of individual genes in each category is listed below the plots.