Recombination of 2Fe-2S Ferredoxins Reveals Differences in the Inheritance of Thermostability and Midpoint Potential

Abstract

Homologous recombination can be used to create enzymes that exhibit distinct activities and stabilities from proteins in nature, allowing researchers to overcome component limitations in synthetic biology. To investigate how recombination affects the physical properties of an oxidoreductase that transfers electrons, we created ferredoxin (Fd) chimeras by recombining distantly-related cyanobacterial and cyanomyophage Fds that present similar midpoint potentials but distinct thermostabilities. Fd chimeras having a wide range of amino acid substitutions retained the ability to coordinate an iron-sulfur cluster, although their thermostabilities varied with the fraction of residues inherited from each parent. The midpoint potentials of chimeric Fds also varied. However, all of the synthetic Fds exhibited midpoint potentials outside of the parental protein range. Each of the chimeric Fds could also be used to build synthetic pathways that support electron transfer between Fd-NADP reductase and sulfite reductase in Escherichia coli, although the chimeric Fds varied in the expression required to support similar levels of cellular electron transfer. These results show how recombination can be used to rapidly diversify the physical properties of protein electron carriers and reveal differences in the inheritance of thermostability and electrochemical properties. Furthermore, they illustrate how electron transfer efficiencies of chimeric Fds can be rapidly evaluated using a synthetic electron transfer pathway..

Laboratory evolution identifies elongated flavodoxins that support electron transfer to sulfite reductases

“…In prior studies, an Escherichia coli strain (EW11) was engineered so that growth is dependent upon Fd-mediated ET from FNR to SIR 39 . This strain cannot grow when sulfate is the sulfur source due to a sulfur metabolism defect, and growth complementation of this defect has been used to report on electron transfer mediated by diverse Fds, including natural pathways made up of corn FNR, Fd, and SIR 39 as well as synthetic pathways containing non-cognate protein electron carriers 41,[45][46][47][48] . As such, this strain represents a simple approach to assess whether Flds can support electron transfer between FNR to SIR that evolved to couple with Fds.…”

Iron-sulfur protein odyssey: exploring their cluster functional versatility and challenging identification

“…[2Fe-2S] ferredoxins have generally low reduction potentials that range between −500 and −150 mV. 5 , 42 Their clusters reside near the protein surface but are protected from degradation by surrounding hydrophobic residues. 43 The [2Fe-2S] ferredoxins can be grouped into three classes: plant-type, adrenodoxin, and thioredoxin-like ferredoxins, all three groups performing electron transport.…”

Evolving a New Electron Transfer Pathway for Nitrogen Fixation Uncovers an Electron Bifurcating-Like Enzyme Involved in Anaerobic Aromatic Compound Degradation

“…In particular, Fds have specialized over evolutionary time to associate with specific partner proteins, allowing Fds to selectively shuttle electrons to specific pathways ( 1 , 2 ). Key factors such as the structure and charge of the Fd binding surface ( 3 ), regulation of Fd abundance ( 4 ), and the reduction potential of the Fd ( 5 – 7 ) affect which partner proteins interact with Fd. In theory, these properties can be altered to enable an Fd to interact with a new partner protein(s) to reroute electron flow, but this remains a challenge for rational design since these properties are ill-defined for many Fds.…”