MnPPIX binding and transfer monitored by absorption and MCD spectroscopy and

Figure 8-9B shows the ESI mass spectrum of a solution containing 2 µM holo-IsdA-N mixed with 3 µM apo-IsdC-N. The data show that holo-IsdAN does transfer MnPPIX to IsdC-N. Four

species are present in the charge state spectra: apo/holo-IsdA-N (+7 and +8), apo-IsdC-N (+7 and +8) and holo-IsdC-N (+7 to +9). A slight increase in the charge state envelope of IsdC-N upon binding MnPPIX could indicate a slightly more open conformation. This result is expected as it has been shown that IsdC-N contains a flexible heme pocket and was able to bind Zn(II)PPIX [19]. Deconvolution shows that transfer is not complete, however, when compared with heme binding [18], with all four species (apo-IsdC-N (14442 Da), holo-IsdC-N (15056 Da), apo-IsdA- N (14626 Da) and holo-IsdA-N (15242 Da)) present in solution.

This same reaction was examined using absorption and MCD spectra. Excess apo-IsdC-N was added to MnPPIX bound IsdA-N. The UV-visible absorption and MCD spectra, Figure 8- 10A do change slightly when compared with holo-MnPPIX-IsdA-N. The Soret band centered at 369 nm becomes much more resolved in the MCD spectrum confirming a change in environment of the MnPPIX. We should note that the heme bound spectra of IsdA-N and IsdC-N are also very similar, due to the same ring binding ligand (tyrosine).

The ESI mass spectrum, Figure 8-9C, of a solution containing 5 µM holo-IsdAN mixed with 6.5 µM apo-IsdE showed charge states for four species: apo/holo-IsdA-N (+7 and +8) and apo/holo-IsdE (+11 to +13). Clearly, MnPPIX does transfer from holo-IsdA-N to apo-IsdE. Deconvolution shows that there exists approximately equal concentrations of apo-IsdA-N (14628 Da) and holo-IsdA-N (15242 Da), and also IsdE (apo-IsdE (30289 Da) and holo-IsdE (30904 Da). Therefore, although MnPPIX does transfer it does not transfer completely. Significantly, heme does not transfer from holo-IsdA-N to IsdE [18]. In the case of MnPPIX, it is possible that the transfer takes place via a dissociative/associative mechanism rather than the highly specific protein-protein interactions that are required for the transfer of heme from IsdC-N to IsdE [18].

Absorption and MCD spectra confirm that a change in the MnPPIX take splace when 3 µM holo-IsdA-N is mixed with 6 µM apo-IsdE, Figure 8-10B. MnPPIX transfer from the holo-IsdA- N to apo-IsdE. The Soret band red shifts from 369 to 372 nm while the Q band shoulder blue shifts from 606 to 600 nm. Overall, the spectrum is complicated and not as resolved as for the IsdA-N to IsdC-N transfer. Therefore, it is probable that these spectra represent a mixture of apo/holo-IsdA-N, apo/holo-IsdE and free MnPPIX in solution as shown in the ESI mass spectrum, Figure 8-9C.

Figure 8-10 UV-visible absorption and MCD spectra recorded following reaction of MnPPIX with apo-IsdA-N, apo-IsdC-N and apo-IsdE. A) Reaction of holo-(MnPPIX)-IsdA- N (3 µM) with apo-IsdC-N (6 µM). The UV-visible absorption and MCD spectra change reflecting the formation of holo-IsdC-N. The Soret band centered at 369 nm becomes more resolved. B) MnPPIX transfer from holo-IsdA-N (3 µM) to apo-IsdE (6 µM). The Soret band red shifts from 369 to 372 nm while the Q band shoulder blue shifts from 592 to 587 nm.

The complete transfer of MnPPIX across all three core Isd proteins was monitored by ESI- MS, Figure 8-9D. Holo-(MnPPIX)-IsdA-N (6 µM) was mixed with apo-IsdC-N (3 µM) and apo-IsdE (5 µM). The ESI-mass spectrum shows that all six apo/holo pairs exist in solution meaning that transfer has taken place and is almost complete, as very little apo-IsdE remains in solution. It has been reported that IsdE has the highest heme affinity [13] and therefore, if MnPPIX is labile within the heme pocket of the IsdA-N and IsdC-N, it might preferentially bind to IsdE.

The absorption and MCD spectral data for this same reaction, Figure 8-11, show the results of mixing a solution of 3 µM holo-IsdA-N with 3 µM apo- IsdC-N and 6 µM apo-IsdE. There is further redshifting of the Soret band to 374 from 372 nm. The Q band region is less resolved

with shoulders at 490 nm and a broad MCD band at 619 nm. This suggests the presence of each of the holo-species in solution, IsdA-N, IsdC-N and IsdE and also the free MnPPIX. Evidence for free MnPPIX in solution is shown by the double band at ~619 nm. The 619 nm peak was not prominent in any other MCD spectrum except for free MnPPIX at pH 10 with a peak at 615 nm. With size-exclusion chromatography performed on all holo-protein solutions, free Mn-PPIX in the MCD spectrum leads us to conclude that binding is not very tight and the ring is labile.

Figure 8-11 UV-visible absorption and MCD spectra recorded for the reaction of holo- IsdA-N (3 µM) with apo- IsdC-N (3 µM) and apo-IsdE (6 µM). The Soret band shifts to 374 from 372 nm. The Q band region is less resolved with shoulder peaks at 490 nm and a broad MCD band envelope is recorded at 619 nm. These data suggest a complicated mixture of MnPPIX species.

8.4 Discussion