Heme-based sensing by the mammalian circadian protein CLOCK

Abstract

Heme is emerging as a key player in the synchrony of circadian-coupled transcriptional regulation. Current evidence suggests that levels of circadian-linked transcription are regulated in response to both the availability of intracellular heme and heme-based sensing of carbon monoxide (CO) and possibly nitric oxide (NO). The protein CLOCK is central to the regulation and maintenance of circadian rhythms in mammals. CLOCK comprises two PAS domains, each with a heme binding site. Our studies focus on the functionality of the murine CLOCK PAS-A domain (residues 103-265). We show that CLOCK PAS-A binds iron(III) protoporhyrin IX to form a complex with 1:1 stoichiometry. Optical absorbance and resonance Raman studies reveal that the heme of ferric CLOCK PAS-A is a six-coordinate, low-spin complex whose resonance Raman signature is insensitive to pH over the range of protein stability. Ferrous CLOCK PAS-A is a mixture of five-coordinate, high-spin and six-coordinate, low-spin complexes. Ferrous CLOCK PAS-A forms complexes with CO and NO. Ferric CLOCK PAS-A undergoes reductive nitrosylation in the presence of NO to generate a CLOCK PAS-A-NO, which is a five-coordinate {FeNO}(7) complex. Formation of the highly stable {FeNO}(7) heme complex from either ferrous or ferric heme makes possible the binding of NO at very low concentration, a characteristic of NO sensors. Comparison of the spectroscopic properties and CO-binding kinetics of CLOCK PAS-A with other CO sensor proteins reveals that CLOCK PAS-A exhibits chemical properties consistent with a heme-based gas sensor protein.

Figure 1

Determination of heme:CLOCK PAS–A stoichiometry by spectrophotometric titration. The main panel shows ΔA spectra for each addition of apoCLOCK PAS–A to a solution of Fe^IIIPPIX. A buffered Fe^IIIPPIX solution of the same concentration was used as the reference sample. The ΔA spectra were obtained by digital subtraction of the reference spectrum from that of the titrated sample. Inset A shows a small subset of the titration curves at the indicated wavelengths. The open symbols represent experimental ΔAs plotted as a function of total CLOCK PAS–A concentration. The solid lines were calculated using the results of the global nonlinear least squares fit to a single step binding reaction to yield a 1:1 complex. Inset B shows the calculated speciation plot wherein concentrations of free FePPIX, heme:CLOCK PAS–A and apoCLOCK PAS–A are plotted as a function of total CLOCK PAS–A added during the titration.

Figure 2

UV-visible spectra of ferric (blue), ferrous (red), and ferrous nitrosyl (green) forms of CLOCK PAS–A.

Figure 3

Soret-excited resonance Raman spectra of ferric (blue) and ferrous (red) CLOCK PAS–A. Both samples contained 50 μM protein in 50 mM potassium phosphate, pH 7.8, 50 mM NaCl. Spectra were excited with 413.1 nm emission from a Kr⁺ laser.

Figure 4

High frequency rR spectrum of CLOCK PAS–A–CO obtained with Soret excitation. Protein concentration of 8 μM, 413.1 nm excitation wavelength, 6 mW laser power at sample. Inset: UV-visible absorbance spectrum of CLOCK PAS–A–CO.

Figure 5

Resonance Raman spectra of CLOCK PAS–A–CO and its ¹³CO isotopologue. A) Low frequency window. B) High frequency window. Protein was in 50 mM potassium phosphate buffer, pH 7.8, 50 mM NaCl. Spectra were recorded at room temperature with 413.1 nm excitation.

Figure 6

ν(Fe–CO)/ν(C–O) correlation plot for iron porphyrin carbonyl complexes. CLOCK PAS–A, ; globins, ▽; cytochrome P450s, △; Horseradish peroxidases, ○; CooA, □; PAS domain proteins, ● (listed in Table 2); NPAS2, Pas A and Pas B domains,. The dashed line correlates ν(Fe–CO) with ν(C–O) for 6c FeCO complexes in which the sixth ligand is thiolate or imidazolate; the solid line represents FeCO adducts with histidine having a neutral imidazole side chain as the sixth ligand. References for the PAS domains are listed in Table 2.

Figure 7

CO recombination following flash photolysis using a 5 ns, 532 nm laser pulse from a Q-switched Nd:YAG laser. A) Transient ΔA spectra recorded between 6 μs to 300 ms following photolysis of CLOCK PAS–A–CO. The spectra were accurately modeled with a two-phase recombination of CO with CLOCK PAS–A at 22 °C. Spectra were referenced against the spectrum of CLOCK PAS–A–CO. For the data shown in this figure, [CLOCK PAS–A–CO]=2.5 μM and [CO]=890 μM. B) CO dependence of k_obs1+k_obs2 and k_obs1·k_obs2 used to determine rate constants for the elementary CO recombination steps. C) Calculated speciation plot showing the time evolution of ■, prompt photoproduct; , intermediate; , CO complex. Inset: Concentrations are plotted on an expanded time axis from 0 to 6 ms in order to more clearly show the appearance of the 6c intermediate at the expense of the pentacoordinate photoproduct.

Figure 8

Visible spectra of the intermediates observed in the CO photolysis experiment. A) Calculated visible spectrum of the prompt photoproduct. B) Calculated spectrum of the intermediate that results when a second endogenous ligand binds to the heme. These visible spectra were determined by peak fitting the features of the ΔA component spectra, as shown in the respective insets. ΔA component spectra were obtained from the two-phase global fit of the ΔA spectra in Figure 7A. Original data, red; global fit, black; positive individual Gaussian bands attributable to the intermediates, black; and negative Gaussian bands due to starting CO complex, blue. The peak parameters of width, relative intensity and frequency for the negative features that arise due to disappearance of the CO complex were held constant at their experimentally determined values during the peak fitting. The parameters for the unknown bands of the intermediates were allowed to vary.

Figure 9

Low frequency rR spectra for ¹⁴N and ¹⁵N isotopomers of CLOCK PAS–A–NO. A) Ferrous CLOCK PAS–A + NO. B) Ferric CLOCK PAS–A + NO. Protein was in 100 mM sodium phosphate, pH 7.1, 100 mM NaCl. Spectra were obtained with 406.7 nm excitation (14 mW at the sample). Red spectra represent the products of the reaction with ¹⁴NO; green spectra represent the ¹⁵NO counterparts; the blue traces are the difference spectra generated by digital subtraction of the ¹⁵NO spectra from the respective ¹⁴NO spectra. The broad band at ~440 cm⁻¹ is due to Raman scattering by the glass NMR tube, in which the samples were contained. To avoid the introduction of artifacts into the difference spectra from baseline corrections, no effort was spent to remove this band from the spectra.

Figure 10

Reaction of ferric CLOCK PAS–A with NO yields ferrous CLOCK PAS–A–NO as judged by rR and absorption spectroscopy. A) High-frequency, Soret-excited rR spectra of the isotopologues of CLOCK PAS–A–NO generated by the reaction of ferric CLOCK PAS–A with NO. Spectra were obtained with 406.7 nm excitation and 14 mW power at the sample. The black spectrum was recorded from ferric CLOCK PAS–A; the red spectrum from CLOCK PAS–A–¹⁴NO; the green spectrum from the CLOCK PAS–A–¹⁵NO isotopologue; and the blue line is the difference spectrum generated by digital subtraction of the CLOCK PAS–A–¹⁵NO spectrum from the CLOCK PAS–A–¹⁴NO spectrum. The spectra of products produced from reaction of NO with ferrous CLOCK PAS–A are identical. B) Absorption spectra tracking the reaction of ferric CLOCK PAS–A with NO. Spectra were monitored at 30 second intervals. The protein solution was 100 mM in sodium phosphate and in NaCl, pH 7.1. The reaction is characterized by clean isosbestic behavior at four points in the spectra, 400, 460, 517 and 563 nm.

Figure 11

Plot showing the inverse correlation between the frequencies of ν(Fe–NO) and ν(N–O) for pentacoordinate {FeNO}⁷ porphyrinates. CLOCK PAS–A–NO and NPAS2 bHLH–PAS–A–NO are indicated by a red star and blue circle, respectively. References for protein complexes are listed in Table 3.

Figure 12

PAS–A domain amino acid sequence alignment of mCLOCK with the heme-binding PAS proteins hNPAS2, mPER 1-3, EcDOS, BxRcoM2, RrCooA, and SmFixL, (Swissprot codes: Mouse [m], O08785; human [h] Q99743; Mouse [m], O35973 [1], O054943 [2], O70361 [3]; [Ec], P76129; [Bx], Q131Y4; [Rr], P72322; and [Sm], P10955 respectively. Sequence alignment was generated by CLUSTALW. Conserved residues are blocked and colored in red. Residues proposed to participate in the heme coordination in mCLOCK based on comparison with hNPAS2 are labeled with a red dot. Proposed heme ligand for ferric mPER2 is Cys215. Heme ligation for ferric SmFixL and EcDos is His194 and His77, respectively. For BxRcoM–1, His70 is involved in heme coordination.

Figure 13

Cross-eyed stereo cartoon representations of the CLOCK PAS–A structural model based on dPer crystal structure (PDB 1WA9). Top: Homology model with all His and Cys sidechains shown in bold, CPK. Bottom: Same CLOCK PAS–A model structure with the side chains of His144 (amber), His196/Cys195 (magenta) and His163/Cys250 (red) shown in bold. With some conformational rearrangement, either of the exchangable His/Cys pairs could join with His 144 to constitute a redox-coupled exchangeable axial ligand triad.

Scheme 1

Top: Illustration of the biphasic relaxation model used to determine k_obs1 and k_obs2 by global nonlinear least squares analysis. Bottom: Elementary reaction steps involved in the rebinding of CO after photolysis by a 5 ns laser flash. Middle: Expressions relating the observed and elementary rate constants.