The physiological reactions of mitochondria and chloroplasts can be reduced to a series of electron transfers, catalyzed by specific enzymes found within the organelles. Thus, we can study the component processes of photosynthesis and respiration by isolating the organelles and measuring specific enzyme activity associated with that organelle.
Photosynthesis requires two processes which can be functionally dissociated, although they work as a unit within the chloroplast. The first process is known as the light reactions, while the second is known by analogy as the dark reactions. The light reactions are rapid changes in the subatomic arrangements of molecules that ultimately split water in the presence of light (photodissociation). Hydrogen atoms from the water are used to reduce NADP to NADPH, and are then used to reduce CO to CHO.
The photodissociation of water in the presence of chloroplast fragments is known as the Hill Reaction. It results from the physical capture of light quanta (energy) into the electron orbit of chlorophyll molecules and the subsequent transfer of an "excited" electron to the orbit of an adjacent molecule. The end products of this reaction are free hydrogen, oxygen and electrons. The electrons are utilized for further chemical reduction reactions, the hydrogen becomes the ultimate hydrogen acceptor in the reactions; and oxygen is a by-product.
Figure 8.1 presents the light reactions, along with the redox potentials of the compounds involved. Complete details of the cyclic and noncyclic pathways of the light reactions are beyond the scope of this manual.
For our purposes, however, the Hill reaction can be monitored by the addition of an electron accepter to the system, and one which will more readily accept the electron. If the electron acceptor is a pigment which alters its color when reduced (by gaining an electron), a simple colorimetric analysis can be used to monitor the photodissociation of water. Basically, the reducing power generated by splitting the water can be used to reduce a dye such as 2,6-dichloroindophenol, which is blue when oxidized, and colorless when reduced.
In respiration, a similar type of oxidation/reduction occurs, except that the process is not physical, but chemical (i.e. it is temperature dependent and slower than a physical reaction). Molecules such as succinic acid are oxidized within the Kreb's Cycle by the removal of both a hydrogen atom and a corresponding electron. Succinic acid dehydrogenation produces fumaric acid; the enzyme performing this reaction is succinic dehydrogenase, and the hydrogen with its electron is normally transferred to NAD.
These reactions are associated with the inner membranes of the mitochondria, and specifically to structures known as respiratory particles. Within these fragments of the inner membrane, the electron transport system functions and the hydrogen and its electron are dissociated. The electron passes through a series of respiratory pigments (the cytochromes) and combines with molecular oxygen through the use of the enzyme cytochrome oxidase. The electron transfer system is outlined in Figure 8.2.
The activities of two enzymes, succinic dehydrogenase and cytochrome oxidase, can be monitored in a manner similar to that for the Hill reaction of photosynthesis. A dye intermediate can be introduced to intercept the electron from the ETS. The gain of electrons will reduce the dye which will consequently change color. The color change may be monitored spectrophotometrically.
The dark reactions of photosynthesis behave differently than the light. They are chemical reactions catalyzed by enzymes and are thus slower than the physical reactions of the light reactions. As chemical reactions, they are temperature dependent. The C3 and C4 pathways of the dark reactions are given in Figures Figures 8.3 and 8.4, respectively.
The chemical reactions of respiration are divided into two major pathways, glycolysis and the Kreb's cycle. These pathways are presented in Figure 8.5. Examination of Figures 8.3 through 8.5 will demonstrate many similarities in the biochemistry of energy metabolism, whether within chloroplasts or mitochondria. Note that glycolysis occurs within the cytoplasm of cells whereas Krebs reactions occur within the mitochondrial stroma. C3 metabolism is within a single plast cell with reactions occuring in the cytoplasm as well as within the chloroplast. C4 metabolism is a process that requires several cells, with distinct compartmentalization of function on a tissue level.
Mitochondria and Chloroplasts can be extracted by gentle rupture of the pertinent cells and differential centrifugation in a media designed to maintain the osmotic and functional integrity of the organelles. The activity of the organelles will vary significantly with the source, the age, and such factors as the length of time of storage before extraction. The organelles can be kept for several days in cold storage, but will gradually degenerate and decompose. It is easier to work with isolated intact organelles than directly with quantosomes or oxysomes, which are unstable under laboratory conditions.
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