A redox switch allows binding of Fe(II) and Fe(III) ions in the cyanobacterial iron binding protein FutA fromProchlorococcus

Abstract

The marine cyanobacterium Prochlorococcus is a main contributor to global photosynthesis, whilst being limited by iron availability. Cyanobacterial genomes typically encode two different types of FutA iron binding proteins: periplasmic FutA2 ABC transporter subunits bind ferric (Fe3+), while cytosolic FutA1 binds ferrous (Fe2+). Owing to their small size and their economized genome Prochlorococcus ecotypes typically possess a single futA gene. How the encoded FutA protein might bind different Fe oxidation states was previously unknown. Here we use structural biology techniques at room temperature to probe the dynamic behavior of FutA. Neutron diffraction confirmed four negatively charged tyrosinates, that together with a solvent molecule coordinate iron in trigonal bipyramidal geometry. Positioning of the positively charged Arg103 side chain in the second coordination shell was consistent with an overall charge-neutral ferric binding state in structures determined by neutron diffraction and serial femtosecond crystallography. Conventional rotation X-ray crystallography using a home source revealed X-ray induced photoreduction of the iron center with observation of the ferrous binding state; here, an additional positioning of the Arg203 side chain in the second coordination shell maintained an overall charge neutral ferrous binding site. Room temperature dose series using serial synchrotron crystallography and an XFEL X-ray pump-probe approach capture the transition between ferric and ferrous states, revealing how Arg203 operates as a switch to accommodate the different iron oxidation states. This switching ability of the Prochlorococcus FutA protein may reflect ecological adaptation by genome streamlining and loss of specialized FutA proteins.

Neutron crystallographic refinement with REFMAC5 from the CCP4 suite

“…Prochlorococcus encodes the FutA protein that can accommodate the binding of iron in either its ferric (Fe 3+ ) or ferrous (Fe 2+ ) state. The structure of FutA has recently been determined using a combination of structural biology techniques at room temperature, revealing the redox switch that allows the binding of both iron oxidation states (Bolton et al, 2023).…”

“…The presence of Arg203 in the second coordination shell suggested the possibility of X-ray-induced photoreduction of the iron centre, leading to a ferrous (Fe 2+ ) binding state. To investigate the protonation of active site residues surrounding the iron, the neutron structure of FutA was determined at 2.1 A resolution using 1 H/ 2 H-exchanged crystals, taking advantage of deuterium fraction refinement (Bolton et al, 2023). The Table 4 Selected neutron models and corresponding X-ray reference models for re-refinement within REFMAC5 using external restraints.…”

“…This represents the first neutron structure to be refined using REFMAC5 and deposited within the PDB. Further mechanistic information on FutA is discussed in a separate publication (Bolton et al, 2023).…”