Of central importance to the study of heme proteins are the effects imposed by axial ligand(s) on the heme structure and, therefore, on the overall activity of the protein. In this study, we confirm and extend the spectroscopic characterization of a mutated sperm whale myoglobin in which the proximal Histidine is replaced with a Tyrosine residue (MbH93Y). The MbH93Y, as well as wild-type sperm whale myoglobin and horse erythrocyte catalase (HEC), was purified and characterized by optical absorption and x-ray absorption (XAS) spectroscopies. Optical absorption spectra of HEC and the metmyoglobin, cyanometmyoglobin, reduced, oxy, and carbon-monoxy forms of both sperm whale myoglobin (SWMb) and MbH93Y were identical to previously reported values within the respective errors:. Extended x-ray absorption fine structure (EXAFS) studies revealed that the proximal bond length in MbH93Y was 2.13 ± 0.03 A, compared to 2.14 ± 0.02 A for sperm whale metmyoglobin and 1.90 ± 0.02 A for catalase. Additionally, the sixth coordination site normally occupied in wild type sperm whale metmyoglobin and in catalase at low temperatures was vacant in MbH93Y, a result corroborated by the optical absorption spectra and cyanogen bromide modification of the distal histidine. Measurements were also made on the cyanide complexes of the three proteins as well, among which, (i) the average iron-to-pyrrole nitrogen bond distance for MbH93Y-CN was 1.96 ± 0.015 A compared to 2.00 ± 0.015 A for WT SWMb-CN and HEC-CN and (ii) the proximal bond length in MbH93Y-CN was 2.07 ± 0.02 A, while that of WT SWMb-CN was 2.10 ± 0.02 A and that of HEC-CN was found to be 2.12 ± 0.02 A. Further, upon exposure to 2-molar equivalents of hydrogen peroxide, sperm whale myoglobin formed a Compound II -like spectrum, while the Soret absorbance of MbH93Y was rapidly, significantly, and irreversibly decreased. Furthermore, the dissociation constants for CN- binding to MbH93Y were found to be, on average, approximately three orders of magnitude higher than those of wild-type sperm whale myoglobin and are consistent with the many-fold higher cyanide binding kinetics for wild type, relative to the mutant protein. Finally, the PK. of the mutant was found to be more than three orders of magnitude higher than that of the native protein. Explanations focusing on probable electronic effects of the phenolate oxygen atom in the sperm whale myoglobin pocket are discussed.
| Identifer | oai:union.ndltd.org:UTAHS/oai:digitalcommons.usu.edu:etd-8296 |
| Date | 01 May 1995 |
| Creators | Chen, Michael J. |
| Publisher | DigitalCommons@USU |
| Source Sets | Utah State University |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | All Graduate Theses and Dissertations |
| Rights | Copyright for this work is held by the author. Transmission or reproduction of materials protected by copyright beyond that allowed by fair use requires the written permission of the copyright owners. Works not in the public domain cannot be commercially exploited without permission of the copyright owner. Responsibility for any use rests exclusively with the user. For more information contact digitalcommons@usu.edu. |
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