Site-directed mutagenesis and spectroscopic analyses have been used to identify the axial ligand residues of the HtsA heme iron and to characterize the axial mutants of HtsA. The His229 and Met79 residues were found to be the heme axial ligands in HtsA. The ferrous heme irons of H229A and M79A HtsA mutant proteins are pentacoordinate and in high-spin state, and so is the heme iron of ferric H229A HtsA, whereas the ferric M79A heme iron appears to have hexacoordination with water and histidine axial ligands. The histidine229 side of the HtsA heme is more sterically limited and more important to the heme affinity than the methionine79 side of the HtsA heme. These findings will be important in rationally designing experiments to elucidate the molecular mechanism and structural basis for the rapid, direct heme transfer from Shp to HtsA. In addition, this study provides spectroscopic features of a protein with Metligated pentacoordinate heme iron.
A previous spectroscopic analysis has unveiled that the hexacoordinate HtsA heme iron is ligated to His and Met residues (18
). The identities of these axial ligands have now been experimentally identified as the methionine 79 and histidine 229 residues in this study. This conclusion is first supported by the effects of Ala replacements of the HtsA histidine and methionine residues on UV-Vis spectra of HtsA. The absorption spectrum of ferric H229A HtsA is similar to the spectra of ferric H102M and R98C/H102M cytochrome b562
mutants, which has a methionine axial ligand (32
). This similarity supports that the H229A replacement results in a mutant protein with a methionine ligand. Conversely, the absorption spectrum of M79A is similar to that of aquometHb whose heme iron has a hexacoordination with water and histidine axial ligands (22
), implying that the M79A replacement results in a mutant protein with a histidine axial ligand. Furthermore, both H229A and M79A at the ferrous state lose the features of hexacoordinate, low-spin heme iron with two strong ligands of wild-type HtsA, lacking the dominant α band in the absorption spectra, a feature possessed by deoxyHb whose ferrous heme iron is pentacoordinate. In addition, ferrous H229A and M79A, but not wt HtsA, rapidly form complexes with CO, which have absorption spectra similar with that of HbCO complex.
MCD spectroscopy is particularly useful for providing information on coordination ligands and numbers of unknown hemoproteins by comparing their MCD spectra with those of hemoproteins with known heme binding properties (33
). The MCD spectrum of ferrous M79A HtsA resembles the MCD spectrum of ferrous horseradish peroxidase (25
), which is known to contain pentacoordinate heme iron with a histidine ligand (26
), supporting that the ferrous M79A heme iron has a pentacoordination with a His axial ligand. The MCD spectrum of ferric M79A HtsA is similar to the MCD spectra of native metmyoglobin and a benzohydroxamic acid adduct of ferric horseradish peroxidase, which are high-spin and have hexacoordination with water and histidine ligands (27
), further supporting that the sixth coordination site of the heme iron in oxidized M79A HtsA is occupied by a water molecule. These MCD features of M79A HtsA further support the conclusion that the methionine79 and histidine229 are the axial ligands in HtsA. The RR spectrum of M79A supports this conclusion.
There had been no firmly established examples for hemoproteins with Met-ligated pentacoordination of heme iron. The heme iron of a H102M mutant of cytochrome b562
ligates to a methionine residue, and its other axial ligand cannot be identified (32
); however, whether the heme iron in this protein is pentacoordinate has not been established. The RR analysis of H229A HtsA indicates that the heme iron in oxidized H229A HtsA is pentacoordinate. This conclusion on the coordination of the H229A heme iron is supported by the observations that H229A HtsA has a blue shift and smaller extinction coefficient for the Soret absorption peak compared with those of the wild-type protein and small intensities of the MCD peaks in the Soret band, like those of known proteins with pentacoordinate heme iron, such as horseradish peroxidase, Aplysia myoglobin, cyanogen bromide-modified myoglobin, and H64L and H64V myoglobin (27
). The axial ligand of the pentacoordinate heme iron in H229A HtsA is apparently methionine since the UV-Vis spectrum of H229A HtsA are similar with that of the cytochrome b562
H102M mutant (32
), but not with those of the pentacoordinate heme iron with a His axial ligand. In summary, ferric H229A HtsA has a pentacoordinate, methionine-ligated heme iron with novel MCD and UV-Vis spectral features (), including particularly an absorption peak at ~600 nm.
Exogenous imidazole can readily ligate to the heme iron of M79A HtsA but not to H229A HtsA, indicating that the coordination site is sterically less accessible on the histidine229 side of the bound heme in HtsA than the other side. This assertion is further supported by the observations that cyanide replaces methionine79 but not histidine229 in the wild-type HtsA protein, that the binding of cyanide to H229A HtsA is weaker than the cyanide binding to M79A HtsA, and that the CO binding to ferrous H229A HtsA is slower than the CO binding to ferrous M79A. Furthermore, His229 is more important than methionine79 for the affinity of HtsA for heme. These observations strongly suggest that the methionine79 side of the heme pocket in HtsA is more exposed to the solvent than the histidine229 site.
It is difficult to measure the heme affinity of hemoproteins with extremely high affinity. We have estimated the affinities of Shp and HtsA for heme by measuring rates of heme association to apoShp and apoHtsA and dissociation from their holo-form (8
). There was an error in the analysis for HtsA in which we mistakenly analyzed the partial reaction of holoHtsA with H64Y/V68F apoMb as a complete reaction (thus, the reported Kd
for heme binding to HtsA in reference 8
was just a lower estimation). Although actual affinities cannot be determined in such cases, H64Y/V68F apomyoglobin is still valuable to estimate relative affinities of hemoproteins. The heme association constant for H64Y/V68F apomyoglobin is estimated to be 1 × 1012
at pH 7.0 based on a bimolecular rate constant of 1 × 108
for hemin association to H64Y/V68F apomyoglobin (35
) and a dissociation rate constant of 1.1 × 10−5
for this double mutant (21
). Based on the spectra of their reaction mixtures with H64Y/V68F apomyoglobin, wild-type, M79A, and H229A HtsA proteins appear to have association constants for heme binding of > 1012
The methionine79 and histidine229 axial residues of HtsA are corresponding to the methionine78 and histidine229 axial ligands of S. aureus
). IsdE is also the lipoprotein component of the ABC transporter and shares 39% amino acid sequence identity with HtsA. HtsA directly and rapidly acquires heme from the surface protein Shp (8
), and IsdE directly but slowly extracts heme from the surface protein IsdC (10
). The histidine229 side of the IsdE heme pocket is also more buried than the methionine78 side (16
). However, there are some differences between these two proteins. Prepared IsdE is a mixture of ferric and ferrous forms (36
), whereas purified HtsA is in the ferric form. H229A IsdE is in a low-spin hexacoordinate complex, which was proposed to be a NO complex, and cannot interact with cyanide (36
). H229A HtsA is in a high-spin state and can form a low-spin complex with cyanide. These differences might be due to the different preparation methods but more likely imply difference in heme binding in these proteins. Such implication is supported by an observation that IsdE cannot acquire heme from Shp nor does HtsA extract heme from IsdC (B. Lei, unpublished data).