The Heme-Based Oxygen Sensor Rhizobium etli FixL: Influence of Auxiliary Ligands on Heme Redox Potential and Implications on the Enzyme Activity

Abstract

Conformational changes associated to sensing mechanisms of heme-based protein sensors are a key molecular event that seems to modulate not only the protein activity but also the potential of the Fe redox couple of the heme domain. In this work, midpoint potentials (E) assigned to the Fe redox couple of the heme domain of FixL from Rhizobium etli (ReFixL) in the unliganded and liganded states were determined by spectroelectrochemistry in the presence of inorganic mediators. In comparison to the unliganded ReFixL protein (+19mV), the binding to ligands that switch off the kinase activity induces a negative shift, i. e. E=-51, -57 and -156mV for O, imidazole and CN, respectively. Upon binding to CO, which does not affect the kinase active, E was observed at +21mV. The potential values observed for Fe of the heme domain of ReFixL upon binding to CO and O do not follow the expected trend based on thermodynamics, assuming that positive potential shift would be expected for ligands that bind to and therefore stabilize the Fe state. Our results suggest that the conformational changes that switch off kinase activity upon O binding have knock-on effects to the local environment of the heme, such as solvent rearrangement, destabilize the Fe state and counterbalances the Fe-stabilizing influence of the O ligand.

A spectroelectrochemical investigation of the heme‐based sensor DevS from Mycobacterium tuberculosis: a redox versus oxygen sensor

“…The high value of E m determined for DevS-CO is consistent with the strong p-acceptor character of CO, which stabilizes the ferrous oxidation state. Curiously, this behavior was quite different for Rhizobium etli FixL (ReFixL), where the value of E m for ReFixL-CO is virtually the same as that determined for the unliganded ReFixL (deoxy-ReFixL) [16]. The spectra obtained at each applied potential for the ligands NO, Imz and CN À are shown in Fig.…”

“…At this point, it is highly relevant to remark that the E m value of DevS is in the region found for other validated oxygen heme-based sensors ( Table 1). Regarding to ReFixL, whose redox potential presented virtually no change upon binding to CO [16], the E m value of DevS-CO is observed at 96 mV, a positive shift of 86 mV in comparison to unliganded deoxy-DevS. Besides the distinct effect of CO on the redox potentials of DevS and FixL, we emphasize their very high CO affinities.…”

“…Despite this difference, the determined E m values are quite similar as seen in Table . As mentioned above, there are several structural and/or electronic aspects that modulate the heme microenvironment thus tuning the redox potential, as described for Re FixL . In fact, as shown in Table , a wide range of redox potentials is reported for heme GAF sensor proteins going from +10 mV (DevS) to −449 mV (All4978) .…”

“…For the spectroelectrochemical studies of DevS in the presence of CN À , the experimental condition was the same as above with the exception of the pH of the solution, which was changed to 9.5 aiming to minimize cyanide escape (as HCN, pK a 9.2) as earlier reported for ReFixL [16]. In this study, upon full oxidation of DevS, a typical spectrum of Fe III -CN DevS (DevS-CN) was obtained with the Soret band at 419 and Q bands at ca.…”

“…In that study, we were also able to show that phosphorylation of DevR, promoted by DevS and DosT, is directly regulated by oxygen binding . Beyond this issue, spectroelectrochemical measurements of a hybrid oxygen heme‐based sensor [nitrogen fixation gene L protein (FixL) from Rhyzobium etli ] showed an interesting correlation trend with the regulated enzymatic activity . That behavior could be linked to significant changes taking place at the heme microenvironment also involving the propionate substituents.…”