Cellular Assays for Ferredoxins: A Strategy for Understanding Electron Flow through Protein Carriers That Link Metabolic Pathways

Abstract

The ferredoxin (Fd) protein family is a structurally diverse group of iron-sulfur proteins that function as electron carriers, linking biochemical pathways important for energy transduction, nutrient assimilation, and primary metabolism. While considerable biochemical information about individual Fd protein electron carriers and their reactions has been acquired, we cannot yet anticipate the proportion of electrons shuttled between different Fd-partner proteins within cells using biochemical parameters that govern electron flow, such as holo-Fd concentration, midpoint potential (driving force), molecular interactions (affinity and kinetics), conformational changes (allostery), and off-pathway electron leakage (chemical oxidation). Herein, we describe functional and structural gaps in our Fd knowledge within the context of a sequence similarity network and phylogenetic tree, and we propose a strategy for improving our understanding of Fd sequence-function relationships. We suggest comparing the functions of divergent Fds within cells whose growth, or other measurable output, requires electron transfer between defined electron donor and acceptor proteins. By comparing Fd-mediated electron transfer with biochemical parameters that govern electron flow, we posit that models that anticipate energy flow across Fd interactomes can be built. This approach is expected to transform our ability to anticipate Fd control over electron flow in cellular settings, an obstacle to the construction of synthetic electron transfer pathways and rational optimization of existing energy-conserving pathways.

Characterization of photosynthetic ferredoxin from the Antarctic alga Chlamydomonas sp. UWO241 reveals novel features of cold adaptation

“…S4). All UWO241 Fds were predicted to contain the [2Fe‐2S] binding domain and clustered within previously defined classes in green algae (Yang et al ., ; Sawyer & Winkler, ) and plants (Fukuyama, ; Atkinson et al ., ) (Fig. ).…”

Genomic Enzymology: Web Tools for Leveraging Protein Family Sequence–Function Space and Genome Context to Discover Novel Functions

“…76 (D) SSN for ferredoxins compared with a phylogenetic tree. 40 (E) SSN for IspH in isoprenoid biosynthesis. 56 (F) SSNs for members of the DRE-TIM metallolyase superfamily.…”

“…56 (F) SSNs for members of the DRE-TIM metallolyase superfamily. 52 Figures reproduced with permission from refs (40), (43), (52), (56), (60), and (76). …”

Photosynthetic fuel for heterologous enzymes: the role of electron carrier proteins

“…Directed evolution-the generation of random or semi-random variants of an enzyme combined with screening of large variant libraries for desirable parameters (Dalby 2014)-might be well suited to tackle this task. However, this approach is contingent on the development of high-throughput methods to screen ferredoxin-dependent enzyme activity, approaches to which were recently proposed (Atkinson et al 2016).…”