Analysis of the L116K Variant of CooA, the Heme-containing CO Sensor, Suggests the Presence of an Unusual Heme Ligand Resulting in Novel Activity

Youn, Hwan; Kerby, Robert L.; Thorsteinsson, Marc V.; Clark, Robert W.; Burstyn, Judith N.; Roberts, Gary P.

doi:10.1074/jbc.m203684200

Cited by 20 publications

(26 citation statements)

References 23 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…(It is unknown why the same I113D CooA variant accumulates poorly hemecontaining CooA in 1ϫ MOPS-buffered media anaerobically in the presence of CO.) Originally, we planned to purify a CooA variant with hydrophilic residues at position 116 as well, but we failed because such variants were unstable. Instability of heme-containing CooA variant at this position has been already reported (23). Fig.…”

Section: Weakening Thementioning

confidence: 81%

“…L116H CooA is intriguing because His could be a ligand in the Fe(II) form, as has strongly been suggested for Lys in L116K CooA (23). We previously showed that Lys ligation in this variant led to the unusually high activity of Fe(II) form and decrease of its activity upon CO binding (23). It is a reasonable hypothesis that His 116 ligation could be the origin for this phenotype in L116H CooA.…”

Section: Weakening Thementioning

confidence: 99%

“…L116R CooA accumulates heme-containing protein at negligible levels, so its inactivity probably reflects an absence of that species, as discussed below. L116H CooA is intriguing because His could be a ligand in the Fe(II) form, as has strongly been suggested for Lys in L116K CooA (23). We previously showed that Lys ligation in this variant led to the unusually high activity of Fe(II) form and decrease of its activity upon CO binding (23).…”

Section: Weakening Thementioning

confidence: 99%

See 2 more Smart Citations

The Role of the Hydrophobic Distal Heme Pocket of CooA in Ligand Sensing and Response

Youn

Kerby

Roberts

2003

Journal of Biological Chemistry

Self Cite

View full text Add to dashboard Cite

CooA from Rhodospirillum rubrum is a heme-containing transcriptional activator that becomes activated only upon binding CO. The basis for this specificity has been probed in a CooA variant, termed ⌬P3R4 CooA, lacking two residues adjacent to the Pro 2 heme ligand, which weakens that ligand. ⌬P3R4 CooA can bind imidazole and CN ؊ , as well as CO, and form a 6-coordinate low spin adduct with each. However, in contrast to the case with CO, imidazole and CN ؊ do not stimulate the DNA binding activity of ⌬P3R4 CooA. This result indicates that the CO-specific activation of CooA is not merely the result of creation of a 6-coordinate CooA adduct but that there must be another element to this response. One feature of CooA activation is modest repositioning of the C-helices upon CO binding, so we altered a portion of the C-helix (residues Ile 113 and Leu 116 ) located near the heme-bound CO in wild type CooA, and we investigated the effect on CO-specific activation. Surprisingly, the sizes of Ile 113 and/or Leu 116 positions are not critical for CooA activation by CO, disproving a precise interaction between these residues and the CO-bound heme as a basis for the CO activation mechanism and CO ligand specificity. In contrast, hydrophobic residues at these positions contribute to the activation. Some CooA variants altered at these positions in the background of ⌬P3R4 were also found to show low but reproducible activation in response to imidazole binding to the heme. A model for the role of hydrophobicity in CooA activation and specificity is suggested.

show abstract

Section: Weakening Thementioning

confidence: 81%

Section: Weakening Thementioning

confidence: 99%

Section: Weakening Thementioning

confidence: 99%

See 1 more Smart Citation

The Role of the Hydrophobic Distal Heme Pocket of CooA in Ligand Sensing and Response

Youn

Kerby

Roberts

2003

Journal of Biological Chemistry

Self Cite

View full text Add to dashboard Cite

show abstract

“…This means that the heme must slide to the C-terminal side on the helix, and this view is consistent with the close proximity of the Leu 116 , Gly 117 , and Leu 120 triad to the CO-bound heme. Leu 116 is reported to be crucial for CooA activity, and the L116K mutant is active in both ferrous and CO-bound states (20). Leu 116 is close to the heme in our model (Fig.…”

Section: Inactive Ferrous Form-inmentioning

confidence: 88%

“…When Leu 116 is replaced by the heme-coordinating lysine residue, CooA is active both in the presence and absence of CO (20). The importance of the nearby Gly 117 to CooA activation has also been stressed (21).…”

mentioning

confidence: 99%

The C-helix in CooA Rolls upon CO Binding to Ferrous Heme

Yamashita¹,

Hoashi²,

Tomisugi

et al. 2004

Journal of Biological Chemistry

View full text Add to dashboard Cite

CooA is a homodimeric transcriptional activator from Rhodospirillum rubrum containing one heme in each subunit. CO binding to the heme in its sensor domain activates CooA, facilitating the binding to DNA by its DNA-binding domain. The C-helix links the two domains and shapes an interface between the subunits. To probe the nature of CO activation, residues at positions 112-121 on the C-helix were replaced by Asn or Gln and their effects were evaluated by resonance Raman spectroscopy and by the measurements of CO binding affinity. The (Fe-CO) stretching Raman line in CO-bound wildtype CooA was up-shifted by 6 cm ؊1 in the L116Q, G117N, and L120Q mutants, indicating unequivocally that these residues are close to the bound CO. Residues Leu 116 and Leu 120 from each subunit form contacts with the corresponding residues in the opposite subunit, enabling hydrophobic interactions in the inactive ferrous form. Thus, in the CO-bound activated form, both C-helices appear to roll to direct these residues toward the heme, forming a hydrophobic pocket for the bound CO. The CO affinity is approximately one order of magnitude higher in the L112Q, I115Q, L116Q, G117N, L120Q, and T121N mutants but reduced in A114N mutant. The variation indicates that these residues are close to the heme in the ferrous and/or CO-bound forms and are responsible for CooA activation. A roll-and-slide mechanism is proposed for CO activation of CooA.

show abstract

Site‐directed spin label electron paramagnetic resonance spectroscopy as a probe of conformational dynamics in the Fe(III) “locked‐off” state of the CO‐sensing transcription factor CooA

et al. 2018

Self Cite

View full text Add to dashboard Cite

The transcriptional activator CooA belongs to the CRP/FNR (cAMP receptor protein/fumarate and nitrate reductase) superfamily of transcriptional regulators and uses heme to sense carbon monoxide (CO). Effector-driven allosteric activation is well understood in CRP, a CooA homologue. A structural allosteric activation model for CooA exists which parallels that of CRP; however, the role of protein dynamics, which is crucial in CRP, is not well understood in CooA. We employed site-directed spin labeling electron paramagnetic resonance spectroscopy to probe CooA motions on the μs-ms timescale. We created a series of Cys substitution variants, each with a cysteine residue introduced into a key functional region of the protein: K26C, E60C, F132C, D134C, and S175C. The heme environment and DNA binding affinity of each variant were comparable to those of wild-type CooA, with the exception of F132C, which displayed reduced DNA binding affinity. This observation confirms a previously hypothesized role for Phe in transmitting the allosteric CO binding signal. Osmolyte perturbation studies of Fe(III) "locked-off" CooA variants labeled with either MTSL or MAL-6 nitroxide spin labels revealed that multicomponent EPR spectra report on conformational flexibility on the μs-ms timescale. Multiple dynamic populations exist at every site examined in the structurally uncharacterized Fe(III) "locked-off" CooA. This observation suggests that, in direct contrast to effector-free CRP, Fe(III) "locked-off" CooA undergoes conformational exchange on the μs-ms timescale. Importantly, we establish MAL-6 as a spin label with a redox-stable linkage that may be utilized to compare conformational dynamics between functional states of CooA.

show abstract

Analysis of the L116K Variant of CooA, the Heme-containing CO Sensor, Suggests the Presence of an Unusual Heme Ligand Resulting in Novel Activity

Abstract: CooA is the CO-sensing transcriptional activator from

Cited by 20 publications

References 23 publications

The Role of the Hydrophobic Distal Heme Pocket of CooA in Ligand Sensing and Response

The Role of the Hydrophobic Distal Heme Pocket of CooA in Ligand Sensing and Response

The C-helix in CooA Rolls upon CO Binding to Ferrous Heme

Site‐directed spin label electron paramagnetic resonance spectroscopy as a probe of conformational dynamics in the Fe(III) “locked‐off” state of the CO‐sensing transcription factor CooA

Contact Info

Product

Resources

About