Idea Transcript
DETERMINATION OF THE ACID DISSOCIATION CONSTANT OF CYTOCHROME B5 REDUCTASE
by Samantha Chong
A Thesis Submitted to the Faculty of The Wilkes Honors College in Partial Fulfillment of the Requirements for the Degree of Bachelor of Arts in Liberal Arts and Sciences with a Concentration in Chemistry and a Minor Concentration in Mathematics
Wilkes Honors College of Florida Atlantic University Jupiter, Florida May 2008
DETERMINATION OF THE ACID DISSOCIATION CONSTANT OF CYTOCHROME B5 REDUCTASE by Samantha Chong This thesis was prepared under the direction of the candidate’s thesis advisors, Dr. Eugene Smith, and Dr. Terje Hoim, and has been approved by the members of their supervisory committee. It was submitted to the faculty of the Honors College and was accepted in partial fulfillment of the requirements for the degree of Bachelor of Arts in Liberal Arts and Sciences.
SUPERVISORY COMMITTEE: ____________________________ Dr. Eugene Smith ____________________________ Dr. Terje Hoim ____________________________ Dr. Michelle Ivey ______________________________ Dr. Jeffery Buller, Dean, Wilkes Honors College ____________ Date
ii
ACKNOWLEDGEMENTS I would like to thank Dr. Eugene Smith of Florida Atlantic University’s Honors College for all of his guidance and support with the experimental work, analysis, and preparation of this thesis. Moreover, I would like to thank Dr. Smith for four years of guiding me on my chosen path in chemistry and for patiently teaching me the basics and supporting me through all aspects of my academic career. I am extremely grateful to have worked and been taught by a professor like Dr. Smith who is genuinely interested in his work and in the future of his students. I would also like to thank Dr. Terje Hoim and Dr. Michelle Ivey for aiding in the editing process of this thesis. Finally, thank you to M. Barber from the USF School of Medicine in Tampa, FL. for the cytochrome b5 reductase used in this study.
iii
ABSTRACT Author:
Samantha Chong
Title:
Determination of the Acid Dissociation Constant of Cytochrome b5 Reductase
Institution:
Wilkes Honors College of Florida Atlantic University
Thesis Advisors:
Dr. Eugene Smith and Dr. Terje Hoim
Degree:
Bachelor of Arts in Liberal Arts and Sciences
Concentration:
Chemistry
Minor Concentration:
Mathematics
Year:
2008
Most living organisms transduce electron transport chains in order to obtain energy. Flavin adenine dinucleotide (FAD) is a common electron transfer cofactor found in electron transport proteins referred to as flavoproteins. In this study, the different ionization and oxidation states of this cofactor found in cytochrome b5 reductase were identified spectroscopically and quantified as a function of solution potential and pH. The large data sets obtained from these experiments were analyzed and the acid dissociation constant for reduced cytochrome b5 reductase was determined.
iv
To Mom and Dad. You never stopped pushing me for excellence, You never stopped guiding me on the right path while letting me be myself, You never stopped supporting me through any and every endeavor, You never stopped believing in me, never, And now you can stop paying, until grad’ school I guess. Thank You. I love you both.
v
Table of Contents Introduction………………………………………………………………………… 1 Flavin Adenine Dinucleotide……………………………………………… 1 Redox and Oxidation States……………………………………………….. 4 Electrochemistry…………………………………………………………… 5 UV-vis Spectroscopy……………………………………………………… 6 Factor Analysis and Malinowski Data……………………………………. 6 Experimental Goal………………………………………………………… 7 Methods…………………………………………………………………………… 9 General Procedure………………………………………………………… 9 Sample Preparation……………………………………………………….. 9 Experimental Methods……………………………………………………..10 Results and Discussion…………………………………………………………….12 Spectra……………………………………………………………………..12 Factor Analysis…………………………………………………………….13 Species Present…………………………………………………………….14 Oxidation…………………………………………………………..15 Reduction…………………………………………………………..16 Concentration Profile………………………………………………………17 Conclusion…………………………………………………………………………18 References………………………………………………………………………….19 Appendix…………………………………………………………………...19 Bibliography………………………………………………………………..25
vi
Tables Residual Standard Deviations, RSD, and the determined number of factors, n…….13 Figures 1Structure of Flavin Adenine Dinucleotide, FAD……………………………………1 2 FAD active site on cytochrome b5 reductase………...……………………………..2 3 Mechanism of FAD reduction to FADH2……….……………………………….…2 4 Structure of FADH2….……………………………………………………………..3 5 Ionization and Oxidation states of FAD……………………………………………5 6 UV-vis spectrum of cytochrome b5 reductase reduction at pH 7.0………….……12 7 Oxidized cytochrome b5 reductase as a function of pH……………………….….15 8 Reduced cytochrome b5 reductase as a function of pH……………………….…..16 9 Concentration profile of different ionization states of reduced cytochrome b5 reductase……………………………………………………………………….…17
vii
Introduction Flavin Adenine Dinucleotide, FAD, is a coenzyme synthesized from riboflavin, or vitamin B12, and is also is a redox cofactor involved in several important reactions in metabolism (see figure 1). FAD is utilized within all mammals and is present within many flavoproteins such as glucose oxidase, and xanthine oxidase in the cellular metabolic cycle, and also in cytochrome b5 reductase, CB5R, a FAD-containing enzyme in the blood that controls the amount of iron in red blood cells, and helps the cells to carry plenty of oxygen (see figure 2). The essential coenzyme FAD is composed of a flavin group (a tricyclic heteronuclear organic ring) and adenosine diphophate (a nucleotide with two phosphate groups). FAD acts as an electron carrier in energy metabolism existing in two different redox states and its biochemical role usually involves changing between these two states. This change of redox states is due to the flavin group. C - Carbon H - Hydrogen N - Nitrogen Na - Sodium O - Oxygen P - Phosphorus
Figure 1. Structure of Flavin Adenine Dinucleotide, FAD
1
Figure 2. The FAD active site on cytochrome b5 reductase
The flavin group is capable of undergoing oxidation-reduction reactions, and can accept either one electron in a two-step process or can accept two electrons at once. FAD’s importance lays in its abilities to form reduced forms of FAD. The reduced forms of FAD are FADH2 and FADH, and the primary sources of reduced FAD in eukaryotic metabolism are the citric acid cycle and the beta-oxidation reaction pathways. FAD is reduced to FADH2, whereby it accepts two hydrogen atoms: H - Hydrogen N - Nitrogen R – Organic group O – Oxygen
FAD + 2H+ + 2e- → FADH2 Figure 3. Mechanism of FAD reduction FADH2
2
FADH2 is produced as a prosthetic group in succinate dehydrogenase, an enzyme involved in the citric acid cycle; and FADH2 is an energy-carrying molecule. The reduced coenzyme can also be used as a substrate for oxidative phosphorylation in the mitochondria. It is also one of the cofactors that can transfer electrons to the electron transfer chain. FADH2 is reoxidized to FAD, which makes it possible to produce two moles of the universal energy carrier ATP.
C - Carbon H - Hydrogen N - Nitrogen O - Oxygen P - Phosphorus
Figure 4. Structure of FADH2
In order to carry out this study it is important to understand all of the analytical and experimental methods used in the process, and how exactly each method is manipulated to work specifically for this research. In identifying the various ionization and oxidation states of FAD it is necessary to understand how redox reactions occur. Electrochemistry, a specific branch of chemistry, is employed to experimentally determine the equilibrium constants for the electron transfers involved in the oxidation and reduction reactions. Following and aiding in this process, UV-vis spectroscopy was
3
used to measure the reduction process as a function of pH, illustrating the reduction through the overall decrease in absorption over time. This helped in obtaining data that could be analyzed by factor analysis, a statistical method that transforms data into linear combinations and identifies the number of unique species present in solution at each stage of the reduction process. Once factor analysis methods were applied to the data sets, a least squares method was used to determine the concentration of the ionization states present and hence the acid dissociation constant, pKa, of reduced CB5R and hence reduced FAD. Redox and Oxidation States Redox, shorthand for reduction/oxidation reaction, describes all chemical reactions in which atoms have their oxidation number (oxidation state) changed. The term redox comes from the two concepts of reduction and oxidation. It can be explained in simple terms:
•
Oxidation describes the loss of electrons by a molecule, atom or ion
•
Reduction describes the gain of electrons by a molecule, atom or ion
However, these descriptions (though sufficient for many purposes) are not truly correct. Oxidation and reduction properly refer to a change in oxidation number and the actual transfer of electrons may never occur. Thus, oxidation is better defined as an increase in oxidation number, and reduction as a decrease in oxidation number. The oxidation cofactor has three oxidation states (oxidized (FAD), semiquinone (FAD-), & hydroquinone (FAD2-)) and three possible ionization states for each oxidized state; hence its mechanism contains nine separate species, as shown in Figure 5. An equilibrium 4
constant, Eo, and an acid dissociation constant, pKa, represent each oxidation and ionization step respectively.
Figure 5. Ionization and Oxidation States of FAD
This model can be used as a preliminary FAD reduction mechanism, as a starting point and determine the equilibrium constants by measuring them at different pH levels. Electrochemistry Electrochemistry is a branch of chemistry that studies chemical reactions which take place in a solution at the interface of an electron conductor and an ionic conductor (the electrolyte), and which involve electron transfer between the electrode and the electrolyte or species in solution. In general, electrochemistry deals with situations where oxidation and reduction reactions are separated in space or time, connected by an external electric circuit to understand each process. Typically, electrochemistry is used to determine the equilibrium constants for electron transfer.
5
UV-vis Spectroscopy Uv-vis spectroscopy, or ultraviolet-visible spectroscopy, involves the absorption of light ranging from 180 – 780 nm by an atom or molecule. The electrons of atoms or molecules are excited to higher energy levels and then release their energy as they fall back to their unexcited or ‘ground’ states. The amount of light absorbed is detected and plotted as absorption versus wavelength (in nm). With molecules, a more complex absorption is apparent than the few frequencies absorbed with atoms. Atoms have a small number of possible energy states for the absorbing particles while molecules have a more complex absorption due to the many possible electronic states of all the atoms present in the molecule. Typically, the Uv-vis spectrum of a molecule is a signature wave (based on analyte solvent and concentration) rather than the several sharp peaks seen in atoms. Uvvis spectroscopy and absorption follows Beer’s Law (A=ΣεbC, where the variables are absorption, extinction coefficient, path length in cm, and concentration respectively) in that absorption is directly proportional to the concentration of the analyte as long as high concentrations do not cause deviation from the linear behavior. Since multiple species are absorbing in the Uv-vis region and Beer’s Law is obeyed, a combined spectrum of constructive interference is produced. Factor Analysis and Malinowski Data Factor Analysis is a multivariate technique for reducing matrices of data to their lowest dimensionality by the use of orthogonal factor space and transformations that yield predictions and recognizable factors. In this study factor analysis is used as a statistical method in which observed data is transformed into linear combinations to identify the number of unique components. Large quantities of data are produced from
6
the spectra and factor analysis can be carried out efficiently using standard factor analytical computer programs such as MATLAB. MATLAB is a high-level technical computing program that is used to process signals and image them using algorithms. It was used to perform the computationally intensive tasks associated with factor analysis. In May 2007, Edmund Malinowski et al. used factor analysis to reveal the pKa of reduced FAD, as well as the reduction pathway, from the spectroelectrochemical reduction of FAD. Their experimental and analytical methods strongly influenced the methods used in this study. Windows factor analysis, wfa, is used to extract the concentration profiles of molecules in order to provide the oxidation and ionization states of the components while principle factor analysis, pfa, is used to determine the number of species present in the mixture at each pH. Experimental Goal Flavin adenine dinucleotide, FAD, is a vital coenzyme present in mammals. A twoelectron acceptor, FAD is reduced to FADH2 and multiple theoretical papers suggest a two electron/two proton process. To probe this mechanism, electrochemistry is used to determine the equilibrium constants for the electron transfers involved in the oxidation and reduction reactions while spectroelectrochemistry is often employed to analyze the structure of molecules based on differences in the absorbance of electromagnetic radiation. UV-vis spectroscopy was used to measure the reduction process and the spectra produced show the specifics of the redox reaction of FAD, illustrating the reduction of FAD over time at various pHs. Factor Analysis is a statistical method that transforms the spectral data into linear combinations. These linear combinations can then be analyzed using principle factor analysis to determine the number of species present in the solution. 7
Further analysis with windows factor analysis can be used to extract concentration profiles providing the oxidation and ionization states of FAD. The pKa value(s) of compounds influence many characteristics of the compound. Specific to this study, the pKa values are of major importance in determining the activity of enzymes and hence how the physical behaviour of the enzymes are affected by ionization. Cytochrome b5 reductase, CB5R, is a FAD-containing enzyme in the blood. It controls the amount of iron in your red blood cells, and helps the cells carry plenty of oxygen. In this study, the factor analysis of spectroelectrochemical data obtained for CB5R was analyzed and the pKa of the reduced state was obtained. The oxidation and ionization mechanism of FAD consists of nine species of FAD. The goal of this research is to spectroscopically identify the different ionization and oxidation states of this cofactor found in cytochrome b5 reductase and quantify them as a function of solution potential and pH. The large data sets obtained from these spectra are then analyzed by factor analysis to determine the value of these equilibrium constants in cytochrome b5 reductase.
8
Methods General Procedure In order to determine these equilibrium constants, the UV-visible spectra of cytochrome b5 reductase was obtained as a function of oxidation state at various pHs. The oxidation state of the cytochrome b5 reductase was established by the oxidation of xanthine by xanthine oxidase. The electrons generated by this oxidation process were used to reduce the FAD containing enzyme. The data collected was analyzed using principal factor analysis to determine the number of different species present (nine possible, as shown earlier). Once the different species were identified, a least squares analysis was used to determine species concentrations at various pHs. Sample Preparation The following solution was mixed in a cuvette in a glove box in order to create the oxygen-free environment needed for the reaction to occur:
• • • • • •
100 µL of 30 µM Xanthine buffer 100 µL pH buffer 38 µL CB5R 3 µL dye 200 µL water 50 µL Xanthine Oxidase (100 x diluted)
The xanthine buffer was prepared by dissolving 15mg of xanthine in 100 µL of 1 M NaOH and diluting to 10 mL with deionized water. The different pH buffers were prepared by adding either HCl or NaOH until the desired pH was reached and diluted to 0.5 L. Xanthine oxidase was kept frozen to ensure optimal enzyme activity and is always added last as it is the catalyst that fuels the reaction. CB5R, the FAD-containing species, 9
was obtained from M. Barber, USF, School of Medicine, Tampa, FL. Inverting the cuvette mixed this solution carefully and quickly and the cuvette was then set in the UV-vis chamber for spectral analysis. Experimental Methods UV-vis spectroscopy was used to collect spectra at regular intervals to view the reaction in real-time. Spectra were taken on an average of one spectrum per minute and most trials ran for approximately 16 hours starting from FAD oxidation ending with FAD reduction. These spectra were then compared against one another and the data put in matrix form. The number of factors and the concentrations of those components were then determined using various techniques in factor analysis and the factors were assigned to the oxidized and reduced states of FAD. Spectroscopy was originally the study of the interaction between radiation and matter as a function of wavelength. Later the concept was expanded greatly to comprise any measurement of a quantity as function of either wavelength or frequency. Thus, it also can refer to interactions with particle radiation or to a response to an alternating field or varying frequency. In this study, spectroscopy is used to analyze the structure of molecules based on the differences in their absorption of electromagnetic radiation. Electromagnetic radiation in the ultraviolet to visible range proves useful in investigating a compound’s electronic structure. When a compound is excited by UV-vis light, the electrons from filled bonding molecular orbitals raise to unfilled antibonding molecular orbitals and then fall back to their ground state and release a different amount of electromagnetic radiation. The spectra are produced from recording the frequency of this
10
electromagnetic radiation absorbed. The typical UV-vis spectrum of FAD has two broad peaks at 370 cm-1 and 450 cm-1.
11
Results and Discussion Spectra The reduction of cytochrome b5 reductase over time was monitored by UVvisible spectroscopy as a function of pH. A representative data set is shown below in figure 6. Principal factor analysis revealed that there were only two species present during the course of the reaction- oxidized and reduced.
Figure 6. UV-vis spectrum of cytochrome b5 reductase reduction at pH 7.0
12
The spectra collected for pHs 6 through 9 are displayed in the appendix. These pHs were chosen because they cover the physiological range, as well as provide additional data points near the equilibrium point of reduced cytochrome b5 reductase. These spectra illustrate the reduction of FAD to a reduced species through the overall decrease in absorption with time. Each of the spectra had to be corrected for baseline shifting due to instrument noise and pH buffering. A simple statistical algorithm allowed us to pass each spectrum through the same baseline point. Factor Analysis Factor analysis was used to express the observed data as linear combinations in an attempt to identify the number of unique components. MATLAB was used to perform the computationally intensive tasks associated with factor analysis, processing signals and imaging them using innovative algorithms. Malinowski’s MATLAB methods were replicated for our purposes and in order to determine the number of factors present in our data at each pH, the standard deviation (STD), real error (RE), and residual standard deviations (RSD) were calculated and analyzed. Table 1 below illustrates the data obtained.
pH 6.2 6.7 7 7.5 8 8.3
Std 5.3 4.9 7.4 8.7 11.2 7.5
RSD 1 RSD 2 RSD 3 62.9 2.9 1.1 59.7 1.9 1.3 69.8 7.4 1.9 67.1 6.2 3.4 81.6 4.8 2.2 55.5 5.8 2.4
n 2 2 2 2 2 2
Table 1. Residual Standard Deviations, RSD, and the determined number of factors, n
13
The number of factors, n, is determined as the point where the RE>STD. The table above shows two factors being detected. Hence, the spectroscopic data and analysis leads us to believe there are two detectable, pH-independent, factors present in the reduced state: FAD and a reduced species. Species Present After analyzing all of our data with factor analysis, two species appeared to be present in solution during each reduction reaction. We have already discussed the three oxidation states of FAD: oxidized FAD, and the reduced FAD2-, and FADH2.Thus, the fact that only two species are present in the reduction process indicates that there are two separate reactions taking place: FAD + 2e- → FAD2FAD + 2e- + 2H+ → FADH2
14
Oxidation In the oxidation process very little variation between pHs is present and a comparison of oxidized cytochrome b5 reductase at various pH revealed a single species (see Figure 7), thus it is concluded that there was only a single ionization state for the oxidized species.
Figure 7. Oxidized cytochrome b5 reductase as a function of pH
15
Reduction A comparison of reduced cytochrome b5 reductase as function of pH is illustrated in Figure 8. Factor analysis of this data revealed that there are two species present, indicating two different ionization states for the reduced state.
Figure 8. Reduced cytochrome b5 reductase as a function of pH
16
Concentration Profile A least squares method was used to determine the concentration of the two ionization states as a function of pH, and the results are shown in Figure 9. The intersection of the two traces reveals a pKa = 7.0 for the reduced species.
Figure 9. Concentration profile of the different ionization states of reduced cytochrome b5 reductase. [FADH−] and [FADH2] are represented by circles and triangles, respectively.
17
Conclusion After our spectroelectrochemical experimentation and factor analysis of the spectral data, it has been found that there is one species present in the oxidized reaction: FAD and two species present in the reduced reaction: FAD2-, and FADH2.Therefore the reaction can be said to proceed under a simultaneous two-electron transfer reaction and the two proton additions are also simultaneously transferred. More importantly the pKa of cytochrome b5 reductase was analyzed and calculated to be 7.0.
18
Appendix pH 6.2 Wavelength vs. Absorbance
19
pH 6.7 Wavelength vs. Absorbance
20
pH 7.0
21
pH 7.5 Wavelength vs. Absorbance
22
pH 8.0 Wavelength vs. Absorbance
23
pH 8.3 Wavelength vs. Absorbance
24
Bibliography Cable, Morgan and Smith, Eugene. 2005. Identifying the n=2 mechanism of FAD through voltammetric simulations. Analytica Chimica Acta 537 299-306.
Combs, Gerald F. 1998. The Vitamins: Fundamental Aspects in Nutrition and Health. 2nd Edition. San Diego: Academic Press.
Malowski, Edmund R. 2002. Factor Analysis in Chemistry. 3rd Edition. New York: John Wiley and Sons, Inc.
Marohnic et al. 2004. Cytochrome b5 Reductase: Role of the si-Face Residues, Proline-92 and Tyrosine-93, in Structure and Catalysis. Archives of Biochemistry and Biophysics 431, 233-244.
Skoog et al. 2000. Analytical Chemistry, An Introduction. 7th Edition. Brooks Cole Thomas Learning.
Smith, Eugene and Whitaker, Graham. 2006. Reduction Mechanism for Flavin Adenine Dinucleotide. Senior Thesis, Wilkes Honors College of FAU.
25