Humboldt State University ® Department of Chemistry

Richard A. Paselk

Chem 438

Introductory Biochemistry

Spring 2010

Lecture Notes: 5 May

© R. Paselk 2006



Look at chloroplast structure [Figure 15.14a & b, p 462]. Note how the thylacoids are stacked, with granal lamellae between stacked thylacoids, and stromal lamellae facing the plastisol (chloroplast matrix) [Figure 15.15, p 463]. The components of the photosynthetic ETS will be differentially distributed between these two lamellae [Figure 15.17, p 464]. Most of protons will be pumped into the lumen of the thylacoid system. (The important thing here is that the protons are being pumped out of the plastisol.) As in mitochondria, can use proton (Chemiosmotic) gradient to make ATP [Figure 15.16, p 463].

The Light Reactions

(Z Scheme)

Let's look a bit at chlorophyll [Figure 15.1, p 445] and its interaction with light. [Figure 15.2, p 446] Find that there are many chlorophylls/active center. That is, only a small portion of the chlorophyll pool is actually involved in using the light energy (1/300 in Chlorella). What do the rest do? Act together as an antenna [Figure 15.3, p 448; 15.7, p 452]. The transfers between molecules take <10-10 sec with an efficiency of >90%. Higher plants also have beta-carotenes [Figure 15.4, p 448] to absorb other light frequencies. Aquatic plants have different dyes, since only blue-green light penetrates to depth.

So what is light energy used for? Look at the "Z" scheme for photosynthetic electron transport. [Figure 15.12, p 458] Note the three major complexes are not directly connected. Like the mitochondrial complexes they are connected by carriers which diffuse between them. [Figure 15.10, p 455] The cytochrome b6/cytochrome f complex is analogous to Complex III in mitochondria: same electron path and a (plastoquinone) Q cycle for proton pumping.

The initial removal of electrons from water to give oxygen uses a manganese complex.

Note the stoichiometry: one O2 : 2 H2O : 4 e- : 8 H+ pumped : 2 ATP : 2 NADPH

The overall proton pumping and ATP synthesis process is summarized in the overhead showing the distribution of the components of the Z scheme [Figure 15.10, p 455].

Cyclic photophosphorylation involves PSI and cytochrome b5f and the Q pool [Figure 15.10, p 455].

The Dark Reactions

(Calvin Cycle)

We have just looked at the so-called Light Reactions of photosynthesis: the process whereby the energy in sunlight is converted by plants into biological useful energy (ATP) and reducing equivalents (NADPH). Now we want to look at how plants use ATP and NADPH to capture ("fix") carbon dioxide and make glucose: the Calvin Cycle.

Most of the reactions of the Calvin Cycle are familiar. Thus on the Calvin Cycle pathway diagram reactions 1-5 (numbered in bold italics) are from Gluconeogenesis/Glycolysis, and reactions 6-8 (numbered in outline font) are from the Pentose Phosphate Shunt, while reactions 9 & 11 are variations of familiar enzyme catalyzed reactions. The only really new reaction is catalyzed by Ribulose-1,5-bis phosphate carboxylase.

Let's begin our look at the Calvin cycle with the first unique reaction of the cycle: the phosphorylation of Ribulose-5-P to Ru-1,5-bis P by Ru-5-P kinase (reaction 9 on the pathway diagram). The strategy here is to create a precursor which is symmetrically phosphorylated so that it can be cleaved into two 3-PGA's.

In the next reaction, catalyzed by Ribulose-1,5-bis phosphate carboxylase (RuBisCo), carbon dioxide is added to the keto-carbon, giving a six carbon sugar which can now be cleaved into the two three carbon PGA's. As shown in the figure, RuBisCo catalyses the addition of a carbon dioxide to ribulose-1,5-bis phosphate, which then splits to give two molecules of 3-PGA:

Note that in the rearrangement resulting with the carboxylation, the final three carbons become essentially a PGA, in particular the keto carbon now has the same oxidation state as an acid carbon (recall that an alcohol elimination involves no formal oxidation, thus a C-C bond is formally the same oxidation as a C-OH bond). Of course the upper portion also now looks about like a PGA too. The attack by water and subsequent cleavage releases the first 3-PGA, leaving a carbanion on the enzyme. The enzyme constrains the addition of a proton to this achiral intermediate to give only the 3-PGA of the proper chirality.

FYI - RUBISCO Structure

The enzyme itself is large (MW= 560,000 daltons), consisting of 8 large (MW= 56,000) catalytic subunits and 8 small (MW= 14,000) regulatory subunits. The eight catalytic subunits form the core of the enzyme, with the interfaces between them forming eight catalytic sites. The enzyme requires magnesium for activity, and there is a copper in each oligomer.

Once 3-PGA is formed the reactions of glycolysis/gluconeogenesis interconvert it to Ga-3-P and F-6-P, intermediates of the pentose-P pathway, and DHAP. These intermediates are then interconverted to reform Ru-5-P. For every three carbon dioxides incorporated by RuBisCo, one extra 3-PGA is formed which can be used for the synthesis of glucose etc. The overall stoichiometry of the cycle is shown on the Calvin Cycle Flow Diagram. This diagram emphasizes recycling vs. incorporation. (packet)

Calvin Cycle regulation: Ribulose-1,5-bis phosphate carboxylase is the flux generating enzyme of the Calvin cycle, and is the main regulatory enzyme of this cycle. Velocity vs [S] plots yield hyperbolic kinetics vs. ribulose-1,5-bis P, but give sigmoidal kinetics vs. carbon dioxide. The activity of the enzyme increases with increasing pH between pH 7 & 9 (H+ is a (-) effector; recall that with illumination the stromal pH increases due to proton pumping by the light reactions). NADPH and Mg2+ are both positive effectors, both increasing with illumination. A switching mechanism turns RuBisCo off in the dark and back on in the presence of light.

FYI - Additional Regulation

A number of additional enzymes of the cycle are regulated in addition to RuBisCo. These enzymes show differences in activity with illumination. In this case the light effects the enzyme via a reduction path leading from the light reactions which provide electrons to reduce a disulfide on thioredoxin, which can in turn reduce disulfides on the enzymes which, for example, increases the rate for F-1,6-bis phosphatase, and changes the substrate specificity for the Ga-3P DH form NADH in the dark to NADPH in the light. Thus the system favors glucose synthesis in the light and glycolysis in the dark!

C4 Plant Photosynthesis

Background on plant strategies under high heat/light low water conditions

C4 Plants: These plants thrive in environments with high temperatures and low humidities where the stomata in the leaves must be closed. Under these circumstances carbon dioxide concentrations fall in the leaves while oxygen rises, favoring photorespiration over photosynthesis and greatly reducing productivity. (In photorespiration oxygen binds competitively with carbon dioxide at the active site of RuBisCo: the net result is that Ru-1,5-bis P is oxidized and energy and carbon are lost instead of gained.) In C4 plants the photosynthesizing cells are protected from the atmosphere by a layer of mesophyll cells. In these cells the PEP carboxylase reaction is used to capture carbon dioxide, with the resulting oxaloacetate carbons transported to the photosynthesizing cell. [Figure 15.26] The first compound incorporating the carbon dioxide thus has four carbons and hence the name (unlike in the Calvin cycle where the first labeled compound, PGA is C3). The carbon dioxide is then released and used in the Calvin cycle. Note that these plants are investing extra energy from ATP to concentrate carbon dioxide. However, they tend to live in high light environments where cyclic Photophosphorylation can be used to make up this extra ATP with little trouble. A variety of transport mechanisms exist in different plant groups. In another mechanism, CAM carbon dioxide is taken up at night and incorporated into malate. The malate is then used the next day to make PEP. Thus the plants can keep their stomata closed during the day.

Pathway Diagrams

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Last modified 5 May 2010