Assignment of Heme Resonances and. High- and Low-Spin Nitrophorin 2 by 1 H and. Order of Heme Methyl Resonances in High- Spin Ferriheme Proteins

Transcription

1 Supplementary Material for: Assignment of Heme Resonances and Determination of the Electronic Structures of High- and Low-Spin Nitrophorin 2 by 1 H and 13 C NMR Spectroscopy: An Explanation of the Order of Heme Methyl Resonances in High- Spin Ferriheme Proteins Tatjana Kh. Shokhireva, Nikolai V. Shokhirev and F. Ann Walker* Department of Chemistry, University of Arizona, Tucson, AZ Assignment of all heme substituent resonances for NP2-NMeIm and NP2-ImH. In Figure 6 is shown the DQF-COSY map for NP2-NMeIm at 30 o C, which reveals three four-spin systems, A (I-II pairs from the HMQC map), B (III-IV pairs from the HMQC map) and C (6.2, 8.2, 10.0, 12.7 ppm), as well as an apparent three-spin system in the upfield region, at 2.8, 0.9, 0.1 ppm, and another 3-spin system consisting of one resonance at 22.6 that is connected to two closely-spaced resonances near 5.6 ppm. Spin systems A and B are thus identified as heme propionate groups, while C is a protein residue, probably the aromatic ring of Phe-66, for which the fifth spin coupled resonance S1

2 is not apparent; the first 3-spin system is likely due to the terminal part of the side chain of Ile-120, while the second is due to Ser-40. We will return to these after establishing the connectivities around the heme. The DQF-COSY map at 30 o C for NP2-ImH is shown in supporting Figure S3, where it is seen that in addition to the NMeIm complex, the ImH complex also shows the 3-spin system of Ser-40, also with widely-spaced geminal β-ch 2 proton signals. In Figure 5 is shown the WEFT-NOESY map for NP2-NMeIm at 10 o C, where it can be seen that the most downfield-shifted heme methyl resonance (at 16 ppm) has NOE cross peaks with propionate group A. This means that this most downfield-shifted heme methyl must be either 5Me or 8Me. The second heme methyl peak, at 14.2 ppm, shares a cross peak at 9.4 ppm that is at an appropriate chemical shift to be a meso-h resonance, which in turn shows a cross peak with the (buried) fourth heme methyl, which is found at 0.2 ppm at this temperature. Thus, these two methyls are 1Me (or 8Me) and 8Me (or 1Me). The buried (fourth) heme methyl also has, in addition to the NOE with the same meso-h resonance as the second heme methyl, dipolar connectivities with propionate group B. This means that the fourth heme methyl peak at 0.2 ppm is 8Me, the second is 1Me, the first is 5Me, and the third is 3Me. The third heme methyl has a cross peak with a β-vinyl group (0.2, 0.9 ppm), which identifies it as 4-vinyl. So at this point we have identified 1Me, 3Me, 4V, 5Me, 6P, 8Me, and δ-meso-h. The order of the heme methyl resonances at 10 o C is 5>1>3>8 for NP2-NMeIm. In the case of NP2-ImH, similar information is obtained from the WEFT-NOESY, Figure S1, HMQC, Figure S2, and DQF-COSY, Figure S3, maps and the results of analysis of these maps are summarized in the assignments in Table 2, which show that S2

3 the order of the heme methyl resonances at all temperatures investigated is 3>5>1>8. Now let us use the NOESY map of NP2-NMeIm, shown in Figure 5, to check the NOE connectivities around the entire heme ring: Starting with the heme methyl signal with the largest hyperfine shift, 5Me has connectivity with β-meso-h (6.3 ppm) and with 6P-H α (14.6 ppm); the latter and its geminal partner (12.6 ppm) connect with 6P-H β ( 1.3, 3.0). β-meso-h also has a dipolar (NOE) cross peak with 4V-H β (0.2,0.9 ppm). 4V-H α also has a cross peak with 3Me (13.2 ppm). The latter has dipolar connectivity with α-meso-h ( 3.2 ppm), which does not connect with the 2V-H β protons (1.8, 0.8 ppm), but these two vinyl-h β have connectivities with 2V-H α (6.8 ppm) and 1Me (14.2 ppm). 1Me connects to δ-meso-h (9.3 ppm), which also connects to 8Me ( 0.2 ppm). 8Me has an NOE with 7P-H α (7.4 ppm). There are strong connectivities between 7P-H α (7.4, 3.6 ppm) and 7P-H β ( 3.5, 4.4 ppm) propionate protons, and between 7P-H α and γ-meso-h ( 4.9 ppm). γ-meso-h has a very intense NOE cross peak with 6P-H α, the latter of which connects with 5Me. With this 5Me connection the circle is closed, and all heme resonances have been assigned. A similar series of assignments can be made for NP2-ImH from the WEFT-NOESY map shown in supporting information Figure S1, and these assignments are summarized in Table 2. The protons of the two propionate chains of NP2-NMeIm give a lot of cross peaks both for the major and minor resonances (~4:1 ratio). This information can be used to make a more detailed assignment of the propionate protons. Several NOESY experiments were run using different mixing times, when all other parameters were kept constant. Primary NOEs were obtained for all geminal protons, for α-propionate Me group, and α-propionate meso-h. The dependence of primary NOE cross peak S3

4 intensities vs. mixing time at 10 o C is shown in Figure S4. For the propionate group at the 6 position, the α-proton which is close to the 5Me group gives a resonance at 12.6 ppm, while the second α-proton (14.6 ppm) is close to γ-meso-h. In the case of the propionate chain at the 7 position, the α-proton which is close to the 8Me group has its NMR resonance at 7.4 ppm, while the second α-proton, at 3.6 ppm, is close to γ-meso-h. All other NOEs between the propionate protons are a result of spin diffusion in these 4- spin systems. The results are summarized in Table 1 for NP2-NMeIm and in Table 2 for NP2-ImH. Figure S1. 1 H 1D and WEFT-NOESY spectrum of NP2-ImH recorded at 500 MHz, 10 o C. Figure S2. 1 H/ 13 C HMQC spectrum of NP2-ImH recorded at 500 MHz, 20 o C. Figure S3. 1 H DQF-COSY spectrum of NP2-ImH recorded at 500 MHz, 30 o C. Figure S4. NOESY cross-peak intensity for NP2-NMeIm (in arbitrary units) versus mixing time, τ mix. S4

5 Figure S1 S5

6 Figure S2 S6

7 Figure S3 S7

8 Figure S4 S8