Prochlorococcus phage ferredoxin: structural characterization and electron transfer to cyanobacterial sulfite reductases

Abstract

Marine cyanobacteria are infected by phages whose genomes encode ferredoxin (Fd) electron carriers. These Fds are thought to redirect the energy harvested from light to phage-encoded oxidoreductases that enhance viral fitness, but it is unclear how the biophysical properties and partner specificities of phage Fds relate to those in photosynthetic organisms. Here, results of a bioinformatics analysis using a sequence similarity network revealed that phage Fds are most closely related to cyanobacterial Fds that transfer electrons from photosystems to oxidoreductases involved in nutrient assimilation. Structural analysis of myovirus P-SSM2 Fd (pssm2-Fd), which infects the cyanobacterium Prochlorococcusmarinus, revealed a high similarity to cyanobacterial Fds (≤ 0.5 Å root-mean-square deviation). Additionally, pssm2-Fd exhibited a low midpoint reduction potential (–336 mV versus standard hydrogen electrode) similar to other photosynthetic Fds, albeit had lower thermostability (Tm = 28°C) than many other Fds. When expressed in an Escherichia coli strain deficient in sulfite assimilation, pssm2-Fd complemented bacterial growth when co-expressed with a P. marinus sulfite reductase, revealing that pssm2-Fd can transfer electrons to a host protein involved in nutrient assimilation. The high structural similarity with cyanobacterial Fds and reactivity with a host sulfite reductase suggest that phage Fds evolved to transfer electrons to cyanobacterial-encoded oxidoreductases.

Recombination of 2Fe-2S ferredoxins reveals differences in the inheritance of thermostability and midpoint potential

“…Many of the native sites that differ in sequence also vary in charge (n=16) and possess functional groups that can form hydrogen bonds (n=8). Even with these differences, the parental proteins contain similar total numbers of hydrogen bonds, with ml-Fd1 having 123 hydrogen bonds and pssm2-Fd having 133 hydrogen bonds 25,27 .…”

“…having Tm values of 28°C and 74°C, respectively 25,26 . To examine how the stabilities of the 2Fe-2S in the chim-Fds relate to the parental proteins, we measured how their ellipticities (427 nm) changed with increasing temperature (Figure 3).…”

“…To test this idea, we created a set of Fd chimeras by recombining homologs from the cyanobacterium Mastigocladus laminosus Fd (ml-Fd1) and a cyanophage PSSM-2 (pssm2-Fd). These Fds were chosen because they have near-identical electrochemical properties (E° ≅ -340 mV), while their melting temperatures (Tm) differ by >40°C [24][25][26] . In each chimera created, we examined iron-sulfur cluster coordination, E°, thermostability, and ET in a synthetic pathway within cells.…”

“…Zea mays FNR (zm-FNR) to Zea mays SIR (zm-SIR) within a synthetic cellular pathway 24,25,34 . In this cellular assay, ET is quantified by monitoring the growth of an Escherichia coli strain (EW11) that cannot grow on medium containing sulfate as the only sulfur source unless a Fd transfers electrons from FNR to SIR 34 .…”

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

“…To test this idea, we created a set of Fd chimeras by recombining homologs from the cyanobacterium Mastigocladus laminosus Fd (ml-Fd1) and a cyanophage PSSM-2 (pssm2-Fd). These Fds were chosen because they have near-identical electrochemical properties (E° ≅ -340 mV), while their melting temperatures (Tm) differ by >40°C [24][25][26] . In each chimera created, we examined iron-sulfur cluster coordination, E°, thermostability, and ET in a synthetic pathway within cells.…”