Advisor(s)

J. Timothy Sage

Contributor(s)

Paul M. Champion, Jeffrey B. Sokoloff

Date of Award

2010

Date Accepted

8-2010

Degree Grantor

Northeastern University

Degree Level

Ph.D.

Degree Name

Doctor of Philosophy

Department or Academic Unit

College of Arts and Sciences. Department of Physics

Keywords

physics, biophysics, DFT, myoglobin, NRVS, nuclear resonance vibrational spectroscopy, protein dynamics, raman

Disciplines

Physics

Abstract

In this thesis, vibrational dynamics of heme proteins are studied with nuclear resonance vibrational spectroscopy (NRVS) and resonance Raman (RR) spectroscopy, with assistance from vibrational predictions and analysis by density functional theory (DFT) computations.

The first chapter is a brief introduction to heme proteins, focusing on myoglobin and to the role of vibrational spectroscopy in the study of heme proteins. It will address the advantages and importance of vibrational spectroscopies as a key to understanding protein dynamics, structures and functions.

Chapter 2 describes the vibrational spectroscopic methods used in the thesis projects. Both the emerging synchrotron-radiation-based NRVS and the traditional resonance Raman spectroscopy will be explained. Application of these vibrational spectroscopies to study the dynamics of heme proteins are the main purpose of Chapter 3, 4, and 5 in this thesis. Cryogenic instrumentations, general methods of protein sample preparation and methods of vibrational predictions and analysis by DFT computations are also addressed.

In Chapter 3, Compound II is an essential intermediate for heme proteins in activation of oxygen. Previous EXAFS measurements have shown that the Fe-O distance in heme compound II is about 1.7 angstrom, suggesting that the iron oxo is double bonded and unprotonated (i.e., Fe(IV)=O). However, recent X-ray diffraction of crystals of heme protein compound II show that the iron-oxo bonds are long enough (~ 1.9 angstrom) to be single bonds thus the iron-oxo groups are protonated (i.e., Fe(IV)-OH). Motivated by the this controversial results on compound II, we visit the subject by studying the vibrational nature of monooxygenated myoglobin derivatives that have a single oxygen atom bound to the heme iron from the distal coordination: Mb compound II (ferryl Mb, also Mb(IV)=O), hydroxymetMb (Mb(III)-OH) and aquometMb (Mb(III)-OH2). DFT computation tells us that if Mb(IV)=O is protonated, its vibrational characteristics resemble those of Mb(III)-OH. We found that the vibrational characteristics of Mb(IV)=O do not feature any of the three protonation signatures that Mb(III)-OH characterizes: (i) ~250 cm-1 down shift of the Fe-O stretching frequency when protonated; (ii) ~30 cm cm-1 split of the in-plane Fe-Npyr/Fe-O tilt modes and (iii) ~14 cm-1 H/D isotope shift of the Fe-O strech mode. Thus the oxo in Mb(IV)=O is not protonated. The long Fe-O bond in structural models derived from X-ray diffraction experiments might be due to the photoreduction of the heme iron by the high flux x-ray beam.

In Chapter 4, we study ferrous nitrosyl myoglobin (MbNO) and address a long time issue of the vibrational mode assignment of the Fe-NO stretching and Fe-N-O bending motions in the protein. Vibrations involving the FeNO fragment appear at ~450cm-1 and ~560cm-1. Vibrational assignments of these two modes have been controversial for the past 30 years. The nonlinear Fe-N-O geometry leads to significant mixing of Fe-NO stretching and Fe-N-O bending contributions to these modes. The FeNO vibration near 450 cm-1 is easily identified in both NRVS and Raman data. NRVS measurements on oriented single crystals of [Fe(TPP)(1-MeIm)(NO)] reveal that the Fe motion is primarily perpendicular to the plane of the porphyrin, as expected for stretching of the Fe-NO bond. Quantitative comparison of isotopic frequency shifts with NRVS data suggests that motion of the nitrosyl N atom accounts for 80% of the mode energy for the mode near 560 cm-1. The Fe amplitude, and consequently the NRVS signal, is thus relatively low for this mode. We conclude that it is mainly an Fe-N-O bending motion.

In Chapter 5, nitrosyl CuB-myoglobins (CuBMbNO) are studied. CuBMb is a mutant of myoglobin in which a non-heme metal-binding site is designed into the distal heme pocket to simulate the active site of heme copper oxidase (for example, cytochrome c oxidase, the terminal enzyme of the mitochondrial respiratory chain). The metals in this site can change the binding properties of ligands to the heme iron and also affect the heme conformation. Previous UV-Vis absorption and EPR studies have found that for CuBMbNO, a Cu(I) in this site weakly perturbs the heme, while Zn(II) actually breaks the Fe-His93 bond, leaving a 5-coordinate (5c) Fe-NO heme structure. NRVS and cryogenic Raman spectra confirm the 5c nature for Zn(II)-CuBMbNO and 6c nature for Cu(I)-CuBMbNO. NRVS data of Zn(II)-CuBMbNO are very similar to that of Fe(PPIX-DME)(NO), a 5c heme model. Cryogenic Raman spectra show that Zn(II)-CuBMbNO resembles the H93G mutant MbNO, where the Fe-His93 link is absent. Further Raman photolysis measurements show that when the NO is photolyzed, the Fe-His stretch mode recovers and appears at ~226 cm-1. With the help of DFT vibrational analysis, we propose that NO binds to the iron from the proximal side, like what happens to cyt c';. Implications for heme copper oxidase and nitric oxide reductase will be discussed.

A brief summary, as well as future opportunity and challenge in NRVS will be presented in Chapter 6.

Document Type

Dissertation

Rights Information

copyright 2010

Rights Holder

Weiqiao Zeng


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