Insights into the Active Site of Coproheme Decarboxylase from Listeria monocytogenes

Coproheme decarboxylases (ChdC) catalyze the hydrogen peroxide-mediated conversion of coproheme to heme b. This work compares the structure and function of wild-type (WT) coproheme decarboxylase from Listeria monocytogenes and its M149A, Q187A, and M149A/Q187A mutants. The UV–vis, resonance Raman, and electron paramagnetic resonance spectroscopies clearly show that the ferric form of the WT protein is a pentacoordinate quantum mechanically mixed-spin state, which is very unusual in biological systems. Exchange of the Met149 residue to Ala dramatically alters the heme coordination, which becomes a 6-coordinate low spin species with the amide nitrogen atom of the Q187 residue bound to the heme iron. The interaction between M149 and propionyl 2 is found to play an important role in keeping the Q187 residue correctly positioned for closure of the distal cavity. This is confirmed by the observation that in the M149A variant two CO conformers are present corresponding to open (A0) and closed (A1) conformations. The CO of the latter species, the only conformer observed in the WT protein, is H-bonded to Q187. In the absence of the Q187 residue or in the adducts of all the heme b forms of ChdC investigated herein (containing vinyls in positions 2 and 4), only the A0 conformer has been found. Moreover, M149 is shown to be involved in the formation of a covalent bond with a vinyl substituent of heme b at excess of hydrogen peroxide.

. UV-Vis absorption and second derivative (D 2 ) spectra (left) and the high frequency region RR spectra (right) of the coproheme complexes with LmChdC WT and its M149A mutant with and without His-Tag. The band wavelengths and frequencies assigned to 5cHS, 5cQS and 6cLS species are indicated in orange, olive green, and magenta, respectively (see text). The spectra have been shifted along the ordinate axis to allow better visualization. Experimental conditions of the RR spectra: 406.7 nm excitation wavelength, laser power at the sample of 5 mW, average of 16 spectra with a 160 min integration time (WT HTag); average of 9 spectra with a 90 min integration time (WT no HTag); average of 3 spectra with a 30 min integration time (M149A no HTag); average of 4 spectra with a 40 min integration time (M149A HTag).
Mutation affects coproheme binding Figure S2. Coproheme binding to the LmChdC variants. Spectral transitions upon binding of 1 µM coproheme (black line) to 3.5 µM LmChdC M149A (A), Q187A (C), and M149A/Q187A (E). The final, coproheme-bound spectra, are depicted in red (after 5s) and intermediate species (after completion of the first binding phase) are shown in green. Experimental time traces followed at 401 nm and single exponential fits representing biphasic coproheme binding to LmChdC M149A (B), Q187A (D) and time traces and fits at 397 nm for M149A/Q187A (F). The insets show the first fast phases.

S3
Wavelength (  Heme b proteins give rise to spectra typical of a main 6cLS species. In the M149A mutant, two different 6cLS forms are observed.

Figure S4. UV-Vis absorption and second derivative (D 2 ) (Panel A) and RR spectra in the high frequency region (Panel B) of the heme b complexes of the WT and its Q187A
, M149A/Q187A, and M19A mutants. The band wavelengths and wavenumbers in magenta indicate the 6cLS species; those in orange and olive green are assigned to minor 5cHS, 5cQS species, respectively. The spectra have been shifted along the ordinate axis to allow better visualization. The 450−700 nm region of the spectra in Panel A is expanded from 6-to 10-fold. Experimental conditions of the RR spectra: 406.7 nm excitation wavelength, laser power at the sample 10 mW, average of 6 spectra with a 30 min integration time (WT), average of 13 spectra with 130 min integration time (Q187A), average of 11 spectra with 110 min integration time (M149A/Q187A) and average of 10 spectra with 100 min integration time (M149A).

S5
The X-band EPR spectrum of the coproheme-WT complex shows the presence of three different species. The most abundant is characterized by g values at 5.90, 5.10, 2.00 (g 12 = 5.50), which confirms the presence of a 5cQS species. The g values of the bands due to the 5cHS, 5cQS, and 6cLS species are coloured in orange, olive green, and magenta, respectively. The spectra have been shifted along the ordinate axis to allow better visualization. Experimental conditions: temperature 10 K, microwave power 2.1 mW, modulation amplitude 10 G. The asterisk at ca. 330 mT indicates an artifact due to a cavity signal. a This band is due to the overlapping contributions of the bands of 5c and 6c HS. The EPR data are in very good agreement with the RR spectra obtained at 80 K. The 80 K RR spectra confirm the presence of a 5cQS species, a 5cHS species, and a 6cLS form for the WT and mainly a 6cLS with only a small amount of 5cHS for the M149 mutant. Figure S6. RR spectra in the high frequency region at 80 K of the coproheme complexes with WT, M149A, and Mb obtained with exc at 356.4 and 406.7 nm. The frequencies of the bands due to the 5cHS, 5cQS, 6cHS and 6cLS species are coloured in orange, olive green, blue, and magenta, respectively. Experimental conditions: (356.4 nm), laser power at the sample 5 mW and average of 7 spectra with 140 min integration time (WT); laser power at the sample 10 mW, average of 12 spectra with 120 min integration time (M149A); (406.7 nm): laser power at the sample 6 mW (WT and M149A), average of 14 spectra with 280 min integration time (WT), 9 spectra with a 135 min integration time (M149A); laser power at the sample 7 mW, average of 7 spectra with 140 min integration time (Mb). The spectra have been shifted along the ordinate axis to allow better visualization.

S7
The CO conformers of the coproheme-complexes have been identified on the basis of the isotope shift of the ν(FeC) and ν(CO) stretching modes in 13 CO versus 12 CO.

S8
Experimental conditions: coproheme and coproheme-Mb: exc 406.7 nm, laser power at the sample 5 mW, average of 6 spectra with 60 min integration time and 12 spectra with 120 min integration time in the low and high frequency regions , respectively ( 12 CO-coproheme), average of 9 spectra with 90 min integration time and 11 spectra with 110 min integration time, in the low and high frequency regions, respectively ( 13 CO-coproheme), average of 4 spectra with 40 min integration time and 10 spectra with 100 min integration time, in the low and high frequency regions, respectively ( 12 CO-Mb), average of 4 spectra with 40 min integration time and 7 spectra with 70 min integration time, in the low and high frequency regions, respectively ( 13 CO-Mb); coproheme-WT and its selected mutants, exc 413.1 nm, laser power at the sample 1-3 mW, average of 28 spectra with 280 min integration time and 22 spectra with 220 min integration time, in the low and high frequency regions, respectively ( 12 CO-WT), average of 8 spectra with 80 min integration time and 15 spectra with 150 min integration time, in the low and high frequency regions, respectively ( 13 CO-WT), average of 6 spectra with 60 min integration time and 18 spectra with 180 min integration time, in the low and high frequency regions, respectively ( 12 CO-M149A), average of 12 spectra with 120 min integration time and 18 spectra with 180 min integration time, in the low and high frequency regions, respectively ( 13 CO-M149A), average of 9 spectra with 90 min integration time and 15 spectra with 150 min integration time, in the low and high frequency regions, respectively ( 12 CO-Q187A), average of 7 spectra with 70 min integration time and 13 spectra with 130 min integration time, in the low and high frequency regions, respectively ( 13 CO-Q187A), average of 6 spectra with 60 min integration time and 15 spectra with 150 min integration time in the low and high frequency regions, respectively ( 12 CO-M149A/Q187A), average of 7 spectra with 70 min integration time and 12 spectra with 120 min integration time, in the low and high frequency regions, respectively ( 13 CO-M149A/Q187A).

S9
The UV-Vis and RR spectra are typical of the heme b-CO complexes. In the RR spectra the ν(FeC) and ν(CO) stretching modes are indicated in red and have been identified on the basis of the isotope shift in 13 CO versus 12 CO ( Figure S9). Due to the high fluorescent background the ν(CO) stretching mode for all the mutants is not clearly defined. The spectra have been shifted along the ordinate axis to allow better visualization. Panel A: the 480−700 nm region is expanded 10-fold. Panel B: RR experimental conditions: exc 413.1 nm, laser power at the sample 1 to 2 mW, average of 12 spectra with 120 min integration time in both the low and high frequency regions (Mb), average of 16 spectra with 160 min integration time and 12 spectra with 120 min integration time in the low and high frequency regions, respectively (WT); average of 11 spectra with 110 min integration time , (M149A), average of 9 spectra with 90 min integration time (M149A/Q187A), average of 12 spectra with 120 min integration time (Q187A).

S10
The CO conformers of the heme b-CO complexes have been identified on the basis of the isotope shift in 13 CO versus 12 CO of the ν(FeC) and ν(CO) stretching modes.
and ν(CO) modes, are indicated in red. The spectra have been shifted along the ordinate axis to allow better visualization. Experimental conditions: exc 413.1 nm, laser power at the sample 1 to 3 mW, average of 16 spectra with 160 min integration time and 12 spectra with 120 min integration time in the low and high frequency regions, respectively ( 12 CO-WT complex), average of 8 spectra with 80 min integration time and 12 spectra with 120 min integration time in the low and high frequency regions, respectively ( 13 CO-WT complex), average of 11 spectra with 110 min integration time and 12 spectra with 84 min integration time in the low and high frequency regions, respectively ( 12 CO-M149A complex), average of 6 spectra with 60 min integration time and 7 spectra with 70 min integration time in the low and high frequency regions, respectively ( 13 CO-M149A complex), average of 12 spectra with 120 min integration time and 20 spectra with 60 min integration time in the low and high frequency regions, respectively ( 12 CO-Q187A complex), and average of 12 spectra with 120 min integration time and 28 spectra with 84 min integration time in the low and high frequency regions, respectively ( 13 CO-Q187A complex), average of 9 spectra with 90 min integration time and 30 spectra with 30 min integration time in the low and high frequency regions, respectively ( 12 CO-M149A/Q187A complex), average of 11 spectra with 110 min integration time and 30 spectra with 60 min integration time in the low and high frequency regions, respectively ( 13 CO-M149A/Q187A complex).