Unusual Dynamics of Ligand Binding to the Heme Domain of the Bacterial CO Sensor Protein RcoM-2

Abstract

The aerobic Gram-negative bacterium Burkholderia xenovorans expresses two highly homologous carbon monoxide (CO)-responsive transcriptional regulators, RcoM-1 and RcoM-2, which display extraordinarily high CO affinities, even under oxygenic conditions. To gain insight into the origin and perspectives of this feature, we characterized the ligand-binding properties of the N-terminal, heme-binding Per/Arnt/Sim sensor domain of RcoM-2 by time-resolved spectroscopy. We show that upon photodissociation of the heme-ligand bond, CO geminately rebinds to the heme with picosecond time constants and more than 99% rebinding yield, an unprecedented property of native heme proteins. Remarkably, the rebinding kinetics speeds up when the protein motions are slowed by cooling or solvent viscosity. This indicates that the origin of the observed efficient rebinding is a protein-imposed CO configuration in the heme pocket that is highly favorable for binding, a feature strongly in contrast to that of hemoglobins. The binding of CO to the ferrous heme from the solvent requires dissociation of the methionine axial heme ligand. From the kinetics of ligand binding and the extreme stability of the CO complex, we deduce that the dissociation constant for CO is lower than 100 pM. Finally, we show that when the ferric complex is exposed to CO gas or a CO-releasing molecule under oxygenic conditions formation of the ferrous carbonyl complex can occur on a time scale of minutes in the presence of a redox mediator. These findings pave the way for possible applications of the RcoM-2 heme domain as a CO sensor and/or scavenger.

Ultrafast CO Kinetics in Heme Proteins: Adiabatic Ligand Binding and Heavy Atom Tunneling

“…Non-exponential behavior and slowing down of ultrafast CO rebinding kinetics over a narrow range of increasing temperature has also been reported in other heme systems such as DNR, RrCooA and DosT 30 as well as for RcoM-2 31 . However, it has been suggested that the CO rebinding to these proteins involves no enthalpic barrier and the non-exponential behavior is attributed to a distribution of activation entropies 30–31 .…”

“…These and other studies have shown that a wide variety of heme proteins, including CooA proteins 6, 32 , nitrophorin 4 21 , CBS 33 , tr-HBO 34 , RcoM-2 31 and mutants of cytochrome c 35 rebind CO on the sub-nanosecond time scale with high geminate yield. In this report we explore the geminate rebinding kinetics of CO to ChCooA, over a broad temperature range (300 K to 20 K).…”

“…6B). This non-adiabatic scenario is difficult to reconcile with the fact that the iron triplet state has not been detected in these reactions 30–31, 34 .…”

“…Under this condition, the triplet state population should be a detectable intermediate during the ultrafast CO rebinding reaction. However, optical experiments 30–31, 34 reveal no evidence of the S=1 intermediate when the ultrafast rebinding is followed with broad spectral bandwidth. Only the reactant (S=2 quintet state) and the product (S=0 singlet state) along with a clear isosbestic point have been observed 20 .…”

Multiphysics model development to characterize fundamental mechanism of bio-responsive hydrogels

“…In general, free hemoglobin consists of two components, the organic and inorganic parts [96][97][98], where the inorganic heme iron complex of hemoglobin binds with oxygen O2 molecules [99][100][101], forming oxygen-heme iron complexes [102]. Interestingly, the association of oxygen O2 with the iron complex leads to a tense-to-relax structural change in the hemoglobin, altering oxygen O2 affinity of the free hemoglobin [103][104][105][106].…”