The very negative FeIII/II potential of cytochromes P450 relative to other heme proteins has been ascribed to the strong donating character of the axial cysteinate ligand. This effect has been modeled in cytochrome c: substitution of the native axial methionine for cysteine decreases the FeIII/II potential by an impressive 652 mV, from 262 vs SHE for the Met/His ligated variant to -390 mV vs SHE for the Cys/His variant (31). Even within cytochrome P450, the reduction potential can be further reduced by increasing the electron donating character of the cysteinate ligand. Removal of a single amide proton proposed to stabilize the cysteinate negative charge shifts the FeIII/II potential negative by
In order to facillitate cyclopropanation activity in vivo, it was necessary to shift the reduction potential sufficiently positive to allow reduction by NADPH. In cytochrome c, it was observed that axial ligation by a weakly donating water molecule raises the reduction potential of the His/H2O ligated variant (FeIII/II: -45 mV vs SHE) by 345 mV compared to the Cys/His variant (31). To that end, we hypothesized in P450, substitution of the cysteinate axial ligand with the weakly donating serine alcohol would be expected to shift the C400S reduction potential positive compared to wild-type. The pKa of serine (~15) is approximately 7 pH units above that of cysteine (~8), and so while cysteine remains deprotonated as the cysteinate ligand in both ferric and ferrous states of the enzyme, we anticipated that serine would remain protonated in at least the ferrous form.
The serine-ligated mammalian P450 mutant has been suggested to be serinate in the ferric form, and serine in the ferrous form based on analysis of absorption spectra and magnetic circular dichroism (16), and we anticipated that similar ligation in Ser-P450-BM3 would result in a more positive FeIII/II potential.
V. Physical Characterization of ABC-CIS V.I. X-Ray Crystallography Statistics
Figure S1. Active site and protein alignments of BM3-CIS with ABC-CIS and wild type P450BM3.
To investigate the nature of enhanced stereoselectivity in ABC-CIS, crystal structures of both proteins were determined to assess any structural changes that may have occurred due to the axial Cys Ser mutation. The top panels shows alignments of BM3-CIS (green) and ABC-CIS (peach) with left, middle, and right panels showing active site residues, the active site I-helix, and global protein fold, respectively. No significant structural changes were observed (RMSD 0.52 Å). Middle panels: Large variations are observed upon comparing BM3-CIS with the open (ligand-free) form of
rearrangements are observed in active site side chain residues (left) as well as rotations within the I-helix. Global movements are also observed in the N-terminal beta domain as well as F- and G-helices (right, marked by double-headed arrows). These movements are consistent with well-known transitions that occur upon substrate binding and are important for native monooxygenation catalysis. Bottom panels: Alignment of BM3-CIS with a ligand-bound BM3 structure (cyan, taken from PDB# 1JPZ, RMSD 0.52 Å) demonstrates that BM3-CIS and ABC-CIS mimic the closed protein conformation even in the absence of substrate.
Table S1. Data collection and refinement statistics for P450BM3 crystals
BM3-CIS ABC-CIS
pdb accession # 4H23 4H24
Data collection*
Space group I 1 2 1 P 2 21 21
wavelength 1.033 1.033
Cell dimensions
a, b, c (Å) 187.79, 62.74, 210.28 63.16, 124.46, 127.69 α, β, γ (°) 90.00, 115.75, 90.00 90.00, 90.00, 90.00 Resolution (Å) 48.6 - 2.5 (2.5 - 2.6)
**
44.9 – 3.3 (3.3 – 3.5)
**
Rmerge 5.3(39.5) 17.6(51.4)
I / σI 13.4(3.0) 11.8(5.7)
Completeness (%)
98.7(99.2) 99.9(99.9)
Redundancy 2.6(2.6) 5.3(5.4)
Refinement
Resolution (Å) 48.6 - 2.5 44.9 – 3.3
No. reflections 72085 14884
Rwork / Rfree 0.19 / 0.25 0.18/0.26 No. atoms
Protein 14401 6890
Ligand/ion 128 86
Water 196 24
B-factors
Protein 33.9 25.4
Ligand/ion 25.4 19.9
Water 26.7 18.9
R.m.s. deviations
Bond lengths (Å) 0.017 0.016
Bond angles (°) Ramachandran
outliers***
1.70 0.3%
1.65 0.7%
*All data sets were collected from single crystals.
**Highest-resolution shell is shown in parentheses.
*** Ramachandran outliers lie in regions of protein that are known to be flexible and
Fig. S2. Heme electron density of ABC-CIS: Maximum likelihood weighted electron density maps of serine-ligated heme in ABC-CIS. (A) Stereo image of heme bound to ABC-CIS viewed from the top of the heme in the active site. (B) Stereo image of heme bound to ABC-CIS rotated ~90° from panel A shows clear indication of heme-iron ligation by the side chain hydroxyl of Cys400Ser. All atoms are shown as sticks. All electron density maps were contoured at σ = 1.0. For perspective, in panel B, the main chain atoms of residues 399 and 401 are also shown as sticks.
V.II. UV-vis Spectroscopy
Figure S3. Absolute spectra for ferric (blue), dithionite reduced ferrous (red) and carbon monoxide bound ferrous (green) ABC-CISheme. Soret bands (nm): FeIII, 404; FeII, 410, 425; FeII-CO, 411. FeII-CO displays α and β bands at 532 and 565 nm. Insert shows the carbon monoxide ferrous (pink) and the dithionite reduced ferrous (yellow) enzymes at 4.5 µM protein concentration.
The shoulder at 410 nm for the ferrous spectrum is due to incomplete reduction to FeII under the aerobic conditions in which these spectra were taken. Reduction of cytochrome P411 under strict anaerobic conditions, as is the case for the redox titrations (figures S7 and S9) gives a single peak at 421 nm.
Figure S4. Absolute spectra for ferric (blue), dithionite reduced ferrous (red) and carbon monoxide bound ferrous (green) ABC-CISholo. Soret bands (nm): FeIII, 404; FeII, 410, 422; FeII-CO, 411. FeII-CO displays α and β bands at 533 and 566 nm. Ferric spectrum displays a broad peak at 465 nm.
Figure S5. Difference spectra for ferrous carbonyl with respect to ferrous for (A) ABC- CISheme and (B) ABC-CISholo.
Table S2: Comparison of λmax for ABC-CISheme and CYP2B4-C436S (15)
ABC-CISheme
(nm) CYP2B4-C436S (nm)
Ferric resting state 404 405
Ferrous 425 422
Ferrous-‐CO 411 413
-‐0.05 0 0.05 0.1 0.15 0.2 0.25 0.3
350 400 450 500 550 600 650 700