Engineering a highly selective, hemoprotein-based scavenger as a carbon monoxide poisoning antidote with no hypertensive effect

Abstract

Carbon monoxide (CO) poisoning causes 50,000 to 100,000 emergency department visits and ~1,500 deaths in the United States annually. Current treatments are limited to supplemental and/or hyperbaric oxygen to accelerate CO elimination. Even with oxygen therapy, nearly half of CO poisoning survivors suffer long-term cardiac and neurocognitive deficits related to slow CO clearance, highlighting a need for point of care antidotal therapies. Given the natural interaction between CO and ferrous heme, we hypothesized that the hemoprotein RcoM, a transcriptional regulator of microbial CO metabolism, would make an ideal platform for CO-selective scavenging from endogenous hemoproteins. We engineered an RcoM truncate (RcoM-HBD-CCC) that exhibits high CO affinity (K_a,CO = 2.8 × 10¹⁰ M^-1), remarkable selectivity for CO over oxygen (K_a,O2 = 1.4 × 10⁵ M^-1; K_a,CO/K_a,O2 = 1.9 × 10⁵), thermal stability (T_m = 72 °C), and slow autoxidation rate (k_ox = 1.1 h^-1). In a murine model of acute CO poisoning, infused RcoM-HBD-CCC accelerated CO clearance from hemoglobin in red blood cells (RBCs) and was rapidly excreted in urine. Moreover, infused RcoM-HBD-CCC elicited minimal hypertension in mice compared to infused globins (hemoglobin, myoglobin, and neuroglobin), attributed to a comparatively limited reactivity toward nitric oxide (NO) via dioxygenation [k_NOD(RcoM) = 6 to 8 × 10⁶ M^-1s^-1 vs k_NOD(Hb) = 6 to 8 × 10⁷ M^-1s^-1]. These data suggest that RcoM-HBD-CCC is a safe, selective, and efficacious CO scavenger. By limiting hypertension through minimal NO scavenging, RcoM-HBD-CCC improves end-organ adverse effects compared with other hemoprotein-based therapeutics.

Keywords: carbon monoxide poisoning; hemoprotein; nitric oxide; therapeutic.

Figure 1.

RcoM-HBD-CCC spectroscopic and stability properties All measurements carried out in phosphate buffered saline (PBS, 10 mM, pH 7.4). (A) Heme coordination environment for RcoM-HBD-CCC. (B) Electronic absorption (UV-Vis) spectra for RcoM-HBD-CCC heme species. (C) Thermal unfolding of RcoM-HBD-CCC bearing Fe(III) heme. Inset: Decrease in absorbance at 413 nm, corresponding to dissociation of Fe(III) heme from the protein. The melting curve was fit using the Santoro-Bolen equation (red line). (D) Comparison of purified RcoM-HBD-CCC spectra before and after chemical oxidation with excess potassium ferricyanide. Top: Reduction of heme using sodium dithionite prior to chemical oxidation yields an admixture of unliganded Fe(II) and Fe(II)-CO species. Bottom: Reduction of heme using sodium dithionite after chemical oxidation yields pure unliganded Fe(II) species. (E) Autoxidation kinetics for Fe(II)-O₂ RcoM-HBD-CCC heme under aerobic conditions at 37 °C. Inset: Kinetic traces were fit to single exponential decay functions to determine the observed rate of autoxidation, k_autox = 1.1 h⁻¹ (red lines).

Figure 2.

Determination of kinetic and thermodynamic parameters for CO binding to RcoM-HBD-CCC. (A) (Left) Spectral changes in the visible (Q-band) region upon stopped-flow rapid mixing of Fe(II) RcoM-HBD-CCC (10 μM, solid black line) and CO-saturated PBS at 25 °C and 297 μM CO, to yield Fe(II)-CO RcoM-HBD-CCC (solid blue line). (Right) Corresponding kinetic traces following the loss of signal from Fe(II) RcoM-HBD-CCC (absorbance at 564 nm, grey line) and formation of Fe(II)-CO RcoM-HBD-CCC (absorbance at 578 nm, red line) at 25 °C. Black dashed lines depict best fits to a single exponential function. (B) Plot of observed pseudo first-order rate constants for CO binding (k_obs) to Fe(II) RcoM-HBD-CCC as a function of CO concentration at 25 °C. Each data point represents the average observed rate constant for four separate mixing events. A simple linear regression yields k_on,CO = 4.67×10⁴ M⁻¹s⁻¹. (C) Kinetics of CO dissociation from Fe(II)-CO RcoM HBD-CCC (6.6 μM) at 25 °C, as measured by replacement with NO over 8 days. Reference data for Fe(II)-CO (blue dashed line) and Fe(II)-NO (black dashed line) RcoM-HBD-CCC species are superimposed over CO displacement data, normalized to highest peak intensity for each respective species. (D) Corresponding kinetic trace of CO dissociation, as measured by change in Soret absorbance at 423 nm as CO is replaced by NO at Fe(II) heme (green circles). The data were fit to a single-exponential decay function (black line) to determine k_on,CO = 1.67×10⁻⁶ s⁻¹. (E) Transient electronic absorption spectra for Fe(II)-CO RcoM-HBD-CCC at different delay times (t) following CO dissociation by flash photolysis. Soret features are plotted as difference spectra where ΔOD(t)=Abs(t)-Abs(t₀) and t₀ is the initial Fe(II)-CO spectrum. (F) CO geminate rebinding kinetics measured at 437 nm following flash photolysis (blue line). The black line represents the curve of best fit to a single exponential function (t_½= 195 ps; τ=281 ps).

Figure 3.

RcoM-HBD-CCC rapidly scavenges CO from RBCs ex vivo under aerobic conditions. CO-saturated murine RBCs (>90% HbCO, 13 μM heme) were incubated with RcoM-HBD-CCC (>99% Fe(II)-O₂, 13 μM heme) at 37 °C for 10 min total. Representative time courses following spectral features for lysed RBCs (A) and cell-free RcoM-HBD-CCC (B). (C) Combined kinetic data summarizing CO transfer from RBC-encapsulated HbCO (red circles) to extracellular RcoM-HBD-CCC (blue squares). Fractions of CO-bound species were determined by spectral deconvolution. Each data point represents the average value from three technical replicates, and error bars denote ± 1 SD. Dashed lines represent best fits to single-exponential functions (k_obs = 0.027 ± 0.003 s⁻¹).

Figure 4.

RcoM-HBD-CCC accelerates CO clearance in a murine model of severe CO poisoning. (A) (left) Experimental scheme for severe CO poisoning mouse model. Red arrows denote 15 uL blood draws. Body temperature was monitored and maintained at 37 °C throughout the course of the experiment. (right) RcoM dosage and animal numbers. (B) Changes in mean arterial pressure (MAP) as a function of time in the severe, nonlethal CO poisoning model. The average starting blood pressure across all treatment groups is depicted by the vertical dashed line at 83.6 mmHg. (C) (left) Comparison of CO clearance, as measured by the difference in $f_{\mathrm{H}\mathrm{b}\mathrm{C}\mathrm{O}}$ before and after infusion, $\mathrm{\Delta }{f}_{\mathrm{H}\mathrm{b}\mathrm{C}\mathrm{O}}$(T2-T1), as a function of scavenger dose. Statistical significance between vehicle and treatment groups was assessed using ordinary one-way ANOVA (*, p<0.05; ***, p<0.001; ****, p<0.0001). (right) Changes in the fraction of circulating HbCO ($f_{\mathrm{H}\mathrm{b}\mathrm{C}\mathrm{O}}$) after CO exposure and delivery of RcoM-HBD-CCC. Values for $f_{\mathrm{H}\mathrm{b}\mathrm{C}\mathrm{O}}$ were quantified by spectroscopic analysis of lysed RBCs isolated from whole blood drawn at four time points throughout the experiment. All $f_{\mathrm{H}\mathrm{b}\mathrm{C}\mathrm{O}}$ values were adjusted to a starting value of 0.76, the average initial $f_{\mathrm{H}\mathrm{b}\mathrm{C}\mathrm{O}}$ at T1 across all treatment groups. (D) Representative spectroscopic data for plasma samples (diluted 40-fold in PBS) taken at different time points following RcoM-HBD-CCC infusion (T2 to T4) in the severe CO poisoning model. Spectra are compared before (solid lines) and after chemical reduction using sodium dithionite (dashed lines) to highlight the degree of CO binding. (E) Assessment of RcoM-HBD-CCC speciation and concentration in plasma. Values for f_CO-RcoM (top) and scavenger concentration (bottom) were quantified by spectroscopic analysis of plasma samples isolated from whole blood. (F) Representative images of urine samples collected 45 min after acute CO exposure and subsequent infusion with scavenger. (G) Spectroscopic analysis of urine samples reveal that RcoM-HBD-CCC scavenger eliminated was >90% CO-bound. For hemodynamic data, error bars denote ± SEM; for all other data, error bars denote ± SD.

Figure 5.

Intravenous infusion of RcoM-HBD-CCC does not elicit hypertension in mice with or without CO exposure. (A) Mean arterial pressure (MAP) as a function of time in the severe, nonlethal CO poisoning model with intravenous infusion of PBS vehicle (n=4, black), StHb at a dose of 800 mg/kg (50 μmol/kg heme; n=3, red), or RcoM-HBD-CCC at a dose of 1020 mg/kg protein (60 μmol/kg heme; n=4, blue). Average starting blood pressure across all treatment groups was 83.6 mmHg (black dashed line). Black arrows denote ten-minute time intervals following CO exposure. (B) Highlighted values for change in MAP (ΔMAP) following the initiation of CO exposure. Each data point represents the difference in blood pressure between the starting value at t=0 and ten-minute time intervals following CO exposure for each replicate. (C) MAP as a function of time in healthy animals after intravenous infusion of PBS vehicle (n=3, black), StHb at a dose of 480 mg/kg protein (30 μmol/kg heme; n=3, red), or RcoM-HBD-CCC at a dose of 510 mg/kg protein (30 μmol/kg heme; n=4, blue). Average starting blood pressure across all treatment groups was 86.0 mmHg (black dashed line). Note that the y-axis range in panel C differs from that in panel A. (D) Highlighted ΔMAP values as in panel B. Statistical significance between vehicle and treatment groups was assessed using one-way ANOVA with multiple comparisons between means in each treatment group (*, p<0.05). (E) Reaction scheme for the estimation of the NO dioxygenation rate constant for RcoM-HBD-CCC (k_NOD,RcoM) through a competition reaction with StHb. (F) Final amount of NO scavenging due to NO dioxygenation by StHb (red bar, 27.4 ± 0.8 nmol) and RcoM-HBD-CCC (blue bar, 2.8 ± 0.1 nmol), as determined by spectral deconvolution (n=4 technical replicates). (G) Correlation between apparent rate of NO dioxygenation (k_NOD) and ΔMAP recorded t = 10 min (3.5 min after complete infusion) in non-CO-poisoned mice. Points represent the mean ΔMAP across biological replicates in each treatment group. Values for k_NOD were derived from the literature for Hb (7 × 10⁷ M⁻¹s⁻¹) and Mb (3.4 × 10⁷ M⁻¹s⁻¹; refs 52 and 53) and from competition kinetics experiments for RcoM-HBD-CCC (7 × 10⁶ M⁻¹s⁻¹) and Ngb-H64Q-CCC (3 × 10⁷ M⁻¹s⁻¹). Note that RcoM-HBD-CCC and StHb (30 μmol/kg heme) were administered at a higher dose than equine myoglobin (Mb) and Ngb-H64Q-CCC (20 μmol/kg heme). For hemodynamic data, error bars denote ± SEM; for all other data, error bars denote ± SD.