Ultrafast Spectroscopy Evidence for Picosecond Ligand Exchange at the Binding Site of a Heme Protein: Heme-Based Sensor YddV

Lambry, Jean−Christophe; Stráňava, Martin; Lobato, Laura; Martínková, Markéta; Shimizu, Tôru; Liebl, Ursula; Vos, Marten H.

doi:10.1021/acs.jpclett.5b02517

Cited by 12 publications

(6 citation statements)

References 34 publications

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“…Despite its function as a heme domain of a CO-activated transcriptional regulator, RcoMH-2 can also bind NO, in the ferrous as well as the ferric (Figure A, inset) state. The latter spectrum, which has not been reported previously for an RcoM protein, displays maxima at 416, 531, and 564 nm, similar to that of other Fe(III)–His–NO complexes. − Figure shows the transient spectra observed upon NO photodissociation and the associated NO recombination kinetics. In both cases, transient spectra reflecting formation of a five-coordinate heme are observed.…”

Section: Resultssupporting

confidence: 79%

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

et al. 2016

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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.

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Section: Resultssupporting

confidence: 79%

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

et al. 2016

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“…To date, GCS proteins with diguanylate cyclase (DGC) output domains have been characterized from E. coli ( Ec DosC), Shewanella putrefaciens (DosD), Bordetella pertussis ( Bpe GReg), Pectobacterium carotovorum ( Pcc GCS), Desulfotalia psychrophila (HemDGC), and Azotobacter vinelandii ( Av GReg) (Burns et al, 2014; Burns et al, 2016; Donné et al, 2016; Kitanishi et al, 2010; Lambry et al, 2016; Nakajima et al, 2012; Sawai et al, 2010; Tagliabue et al, 2010; Tarnawski et al, 2015; Tuckerman et al, 2009; Wan et al, 2009; Wu et al, 2013). All of these DGC-containing GCS proteins have a linking middle domain between the N-terminal sensor globin and C-terminal DGC domain.…”

Section: Diguanylate Cyclase-containing Gcs Proteinsmentioning

confidence: 99%

“…Given the importance of biofilm formation in bacterial growth and survival, including being of great import in infections by various bacterial pathogens, the roles of DGC-containing GCS proteins in controlling O 2 -dependent c-di-GMP production have garnered considerable interest (Donné et al, 2016; Tagliabue et al, 2010; Wu et al, 2013). In addition, as c-di-GMP production can be monitored in vitro , DGC-containing GCS proteins have provided an opportunity to dissect the effects of ligand binding on catalysis, linking these effects to structural rearrangements (Burns et al, 2014; Burns et al, 2016; Lambry et al, 2016; Rivera, Burns, Vansuch, Chica, & Weinert, 2016; Shimizu et al, 2015; Tarnawski et al, 2015; Wu et al, 2013). …”

Section: Diguanylate Cyclase-containing Gcs Proteinsmentioning

confidence: 99%

Mechanism and Role of Globin-Coupled Sensor Signalling

Walker

Rivera

Weinert

2017

Advances in Microbial Physiology

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The discovery of the globin-coupled sensor (GCS) family of haem proteins has provided new insights into signalling proteins and pathways by which organisms sense and respond to changing oxygen levels. GCS proteins consist of a sensor globin domain linked to a variety of output domains, suggesting roles in controlling numerous cellular pathways, and behaviours in response to changing oxygen concentration. Members of this family of proteins have been identified in the genomes of numerous organisms and characterization of GCS with output domains, including methyl accepting chemotaxis proteins, kinases, and diguanylate cyclases, have yielded an understanding of the mechanism by which oxygen controls activity of GCS protein output domains, as well as downstream proteins and pathways regulated by GCS signalling. Future studies will expand our understanding of these proteins both in vitro and in vivo, likely demonstrating broad roles for GCS in controlling oxygen-dependent microbial physiology and phenotypes.

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“…The temporal resolution is determined by the Kerr-gate medium, and was either ~ 200 fs (suprasil, used for C51A, C51A/W214F and C51A/W283F mutants)) or ~ 1 ps (CS 2 , used for C51A/Y343F and C51A/Y343F/ W214F mutants); in the latter case, the signal is higher due to higher efficiency of the medium. Time-resolved absorption experiments were performed as described [15,31], with pump pulses centered at 390 nm. All time-resolved Fig.…”

Section: Methodsmentioning

confidence: 99%

Photochemical processes in flavo-enzymes as a probe for active site dynamics: TrmFO of Thermus thermophilus

Zhuang

Nag

Sournia

et al. 2021

Photochem Photobiol Sci

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Quenching of flavin fluorescence by electron transfer from neighboring aromatic residues is ubiquitous in flavoproteins. Apart from constituting a functional process in specific light-active systems, time-resolved spectral characterization of the process can more generally be employed as a probe for the active site configuration and dynamics. In the C51A variant of the bacterial RNA-transforming flavoenzyme TrmFO from the bacterium Thermus thermophilus, fluorescence is very shortlived (~ 1 ps), and close-by Tyr343 is known to act as the main quencher, as confirmed here by the very similar dynamics observed in protein variants with modified other potential quenchers, Trp283 and Trp214. When Tyr343 is modified to redox-inactive phenylalanine, slower and highly multiphasic kinetics are observed on the picosecond-nanosecond timescale, reflecting heterogeneous electron donor-acceptor configurations. We demonstrate that Trp214, which is located on a potentially functional flexible loop, contributes to electron donor quenching in this variant. Contrasting with observations in other nucleic acid-transforming enzymes, these kinetics are strikingly temperature-independent. This indicates (a) nearbarrierless electron transfer reactions and (b) no exchange between different configurations on the timescale up to at least 2 ns, despite the presumed flexibility of Trp214. Results of extensive molecular dynamics simulations are presented to explain this unexpected finding in terms of slowly exchanging protein configurations.

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Ultrafast Spectroscopy Evidence for Picosecond Ligand Exchange at the Binding Site of a Heme Protein: Heme-Based Sensor YddV

Cited by 12 publications

References 34 publications

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

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

Mechanism and Role of Globin-Coupled Sensor Signalling

Photochemical processes in flavo-enzymes as a probe for active site dynamics: TrmFO of Thermus thermophilus

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