Old Dominion University ODU Digital Commons Chemistry & Biochemistry Theses & Dissertations Chemistry & Biochemistry Fall 2016 Bioenergetics: Experimental Demonstration of Excess Protons and Related Features Haitham A. Saeed Old Dominion University, [email protected] Follow this and additional works at: https://digitalcommons.odu.edu/chemistry_etds Part of the Biochemistry Commons, and the Chemistry Commons Recommended Citation Saeed, Haitham A.. "Bioenergetics: Experimental Demonstration of Excess Protons and Related Features" (2016). Doctor of Philosophy (PhD), Dissertation, Chemistry & Biochemistry, Old Dominion University, DOI: 10.25777/yjam-mg39 https://digitalcommons.odu.edu/chemistry_etds/11 This Dissertation is brought to you for free and open access by the Chemistry & Biochemistry at ODU Digital Commons. It has been accepted for inclusion in Chemistry & Biochemistry Theses & Dissertations by an authorized administrator of ODU Digital Commons. For more information, please contact [email protected]. BIOENERGETICS: EXPERIMENTAL DEMONSTRATION OF EXCESS PROTONS AND RELATED FEATURES by Haitham A. Saeed B.S. August 2007, Alexandria University, Egypt M.Sc. May 2015, Old Dominion University A Dissertation Submitted to the Faculty of Old Dominion University in Partial Fulfillment of the Requirements for the degree of DOCTOR OF PHILOSOPHY CHEMISTRY OLD DOMINION UNIVERSITY December 2016 Approved By: James W. Lee (Director) Richard V. Gregory (Member) Thomas Isenhour (Member) Sandeep Kumar (Member) ABSTRACT BIOENERGETICS: EXPERIMENTAL DEMONSTRATION OF EXCESS PROTONS AND RELATED FEATURES Haitham A. Saeed Old Dominion University, 2016 Director: Dr. James W. Lee Over the last 50 years, ever since the Nobel-prize work of Peter Mitchell’s Chemiosmotic theory, the question whether bioenergetics energy transduction occurs through localized or delocalized protons has been a controversial issue among scientists. Recently, a proton- electrostatics localization hypothesis was formulated which may provide a new and clear understanding of localized and delocalized proton-coupling energy transduction in many biological systems. The aim of this dissertation was to test this new hypothesis. To demonstrate the fundamental behavior of localized protons in a pure water- membrane-water system in relation to the newly derived pmf equation, excess protons and excess hydroxyl anions were generated and their distributions were tested using a proton-sensing aluminum membrane. The proton-sensing film placed at the membrane-water interface displayed dramatic localized proton activity while that placed into the bulk water phase showed no excess proton activity during the entire experiment. These observations clearly match with the prediction from the proton-electrostatics localization hypothesis that excess protons do not stay in water bulk phase; they localize at the water-membrane interface in a manner similar to the behavior of excess electrons in a conductor. In addition, the effect of cations (Na+ and K+) on localized excess protons at the water- membrane interface was tested by measuring the exchange equilibrium constant of Na+ and K+ in exchanging with the electrostatically localized protons at a series of cations concentrations. The equilibrium constant for sodium (Na+) cations to exchange with the electrostatically + localized protons was퐾푃 푁푎determined to be (5.07 ± 0.46) x 10-8 while the equilibrium constant for potassium (K+) cations to exchange with localized protons was determined to + 푃퐾 be (6.93 퐾± 0.91) x 10-8. These results mean that the localized protons at the water-membrane interface are so stable that it requires a ten millions more sodium (or potassium) cations than protons in the bulk liquid phase to even partially delocalize them at the water-membrane interface. This provides a logical experimental support of the proton electrostatic localization hypothesis. One of the basic assumptions of proton-electrostatics localization hypothesis is that it treats liquid water as a proton conductor and that the proton conduction along the water- membrane interface might be a favored pathway for the proton energy coupling bioenergetics across biological membranes. In this study, experimental evidences discussing water acting as a proton conductor were discussed and the conductivity of water with respect to excess protons was estimated. Overall, these findings have significance not only in the science of bioenergetics but also in the fundamental understanding for the importance of water to life in serving as a proton conductor for energy transduction in living organisms. iv © 2016, by Haitham A. Saeed, All Rights Reserved. v This dissertation is dedicated to my parents, my wife and my little daughter Sarah. vi ACKNOWLEDGMENTS First and foremost, I thank God for equipping me to succeed in this endeavor. All praise and glory is to him. I would like to sincerely express my gratitude to everyone who has contributed to this work. I would first like to thank my academic and research advisor Dr. James Lee for his mentorship. I am thankful to him for his academic guidance, patience, and encouragement throughout my research. He has spent countless hours reviewing my work and supporting me in my growth as a researcher. Without his dedication and advice, this dissertation wouldn’t be possible. Secondly, I would like to thank the department of chemistry and biochemistry at ODU for accepting me in the program and providing me with the teaching assistantship during the past five years. I would also like to thank the other members of my dissertation committee, Dr. Isenhour, Dr. Gregory, and Dr. Kumar for their valuable comments and directions. I am grateful to Dr. Gregory for his advice, for allowing me using his equipments to conduct my experiments, and for making valuable contributions to my education at ODU. I would like also to thank Dr. Cooper for his valuable discussions and for providing me with several insights throughout my dissertation. I am exceptionally thankful to the staff members at department of chemistry and biochemistry. Their help and assistance are greatly appreciated. I would like to thank my friends and colleagues who worked closely with me and supported me when needed. I thank Matthew Huff and Cameron Smith for their help and support during these years. Finally, I sincerely express my gratitude to my family members. I thank my parents who encouraged me since I was young to the pursuit of knowledge, investing all that they had to provide me with a good standard education. I also thank my sisters, my wife and my daughter for their faith, support and encouragement in all my endeavors, motives and academic pursuits. vii TABLE OF CONTENTS Page LIST OF TABLES ........................................................................................................................ IX LIST OF FIGURES ................................................................................................................... XIII Chapter 1. INTRODUCTION .................................................................................................................... 1 1.1 BIOENERGETICS, ATP SYNTHESIS AND PROTON GRADIENT ....................... 1 1.2 BACKGROUND INFORMATION .............................................................................. 4 1.3 DISSERTATION HYPOTHESES .............................................................................. 21 1.4 GOALS AND OBJECTIVES OF STUDY ................................................................. 23 2. EXPERIMENTAL DEMONSTRATION OF LOCALIZED EXCESS PROTONS AT A WATER-MEMBRANE INTERFACE ................................................................................... 26 2.1 INTRODUCTION ....................................................................................................... 26 2.2 MATERIALS AND METHODS ................................................................................ 31 2.3 RESULTS AND DISCUSSION ................................................................................. 36 2.4 CONCLUSION ........................................................................................................... 49 3. THE EFFECT OF CATIONS (Na+ and K+) ON LOCALIZED EXCESS PROTONS AT A WATER-MEMBRANE INTERFACE ......................................................................... 50 3.1 INTRODUCTION ....................................................................................................... 50 3.2 MATERIALS AND METHODS ................................................................................ 60 3.3 RESULTS AND DISCUSSION ................................................................................. 64 3.4 CONCLUSION ........................................................................................................... 84 4. EXCESS PROTON CONDUCTION IN PURE WATER ...................................................... 86 4.1 INTRODUCTION ....................................................................................................... 86 4.2 MATERIALS AND METHODS ................................................................................ 89 4.3 RESULTS AND DISCUSSION ................................................................................. 93 4.4 CONCLUSION ......................................................................................................... 108 viii Chapter Page 5. CONCLUSIONS AND FUTURE WORK ........................................................................... 109 5.1 CONCLUSIONS ......................................................................................................