September 11, 1997                                        MAST 634                    Lecture 3: Chemoautotrophy 
 

Important Points from Last Week 

Review of Gibb's Free Energy 
Calculation of changes in potential for redox reactions 
Chemoautotrophy: p 489-495 in Hochachka and Somero on reserve 
Generation of proton-motive force via oxidation of X and reduction of O₂ (O₂ is terminal e^- acceptor) 

How to separate PCR products when they are same site? cloning 

      Useful Web Sites for Illustrations about Cloning and Molecular Biology  

First page with directory: http://www.gene.com/ae/AB/GG/ 
Cloning with plasmids: http://www.gene.com/ae/AB/GG/inserting.html 
http://www.gene.com/ae/AB/GG/plasmid.html 
Restrictionase: http://www.gene.com/ae/AB/GG/restriction.html 
 

      -virtually same for all organisms bacteria to higher plants 

      Chapter 22 in Voet and Voet; p 295-317 in Gottschalk 

      CO₂ is the form of inorganic C used by pathway 

  But different mechanisms for getting CO₂ 

Land plants: CO₂ gas, often not a problem fast-growing plants -- C₄ plants have Hatch - Slack pathway 

Aquatic plants 
      usually not limiting 
      some question about effect of build up in CO₂ conc. on plant growth 

Review of inorganic carbon (DIC)  

      H₂O + CO₂(g) = H₂CO₃ = HCO₃^- + H⁺ = CO₃^2- + 2H⁺ 
                                                 bicarbonate         carbonate 

                at seawater pH, mostly HCO₃^- 

DIC Uptake 

Important to plants and for interpreting ¹³C as paleo-marker - used as index of past CO₂ concentrations 

1. Diffusion of CO₂ 
      - no charge and small, so diffusion is possible 
      - but can be too slow 
2. Active uptake of CO₂ 
3. HCO₃^- uptake 

Carbonic anhydroydrase (CA) mediates this reaction: 

            HCO₃^- + H⁺ = CO₂ + H₂O 

CA is in many cells, such as in gastric mucosa; creates acidity       p 528 in Voet and Voet 

In plants: 

            "Traps" CO₂ in cell, HCO₃^- can't diffuse out as easily 
            Can help create concentration gradient for CO₂ reducing CO₂ concentrations  
 

For HCO₃^- - active uptake, different role for CA; facilitate CO₂ formation 
 

Complex picture; several differences among different plants 

What happens after CO₂ is in cell? 

Dark reactions: Calvin - Bassham - Benson Cycle  
      - found in all autotrophs ---> bacteria to eukaryotes 
      - chemolithotrophics 

Cycle dissected with ¹⁴CO₂ 
      Stable, abundant C = ¹²C 
      radioactive C = ¹⁴C half-life of 5700 yr. 

1. First exp's: add ¹⁴CO₂, look at radioactive organics 
      found 3-P-glycerate (3-PG), suggesting 2-C + ¹⁴CO₂ = 3-PG 
      but never found 2-C sugar 

2. Next; 
      a) Add ¹⁴C ("pulse"); allow plants to take up for some time 
      b) Remove ¹⁴CO₂ 

(Note: This is not a true "pulse-chase" experiment because ¹⁴CO₂ is not chased with nonradioactive (unlabeled or "cold") CO₂, but rather CO₂ is removed completely.) 

 

Data suggest: RuBP + ¹⁴CO₂ 6C ----> 3PG ----> rest of Calvin cycle 

            Consistent with data 
                    - remove of ¹⁴CO₂ ----> more RuBP because it's not building up 
                    - 3PG not endproduct because it is used up 

This reaction is the most important reaction of Calvin Cycle 

Full name for enzyme: RuBP carboxylase/oxygenase 
                    Abbreviations: RuPBcase, Rubisco 
 
Two stages of Calvin Cycle  

      1. 3 RuBP + 3 CO₂ + 9ATP + 6 NADPH= 6 glyceraldhehyde-3-phosphate (abbreviated as GAP, a 3C   sugar) 

      2. Regenerate RuBP 
            5 of 6 GAP ---> RuBP 
            1 of  6 GAP ---> everything else 

     .^.. during stage 2: 5C₃ ---> 3 C₅ 

Overall stoichiometry: 3 CO₂ + 9ATP + 6NADPH ---> GAP + 9 ADP + 8P_i + 6 NADP⁺ 

More details on RuBP carboxylase (RuBPcase) 
      50% of leaf protein ---> 
            RuBPcase 4 x 10⁹ tons /y 
            1 production 11 x 10⁹ 
            Crude oil consumption 3 x 10⁹ 

"Fraction 1" of tobacco = RuBPcase: once thought as possible food source? 

Structure of RuBPcase  
 

Mechanism of CO₂ fixation: see handout; nucleophilic attack 

Other carboxylations 

ß carboxylation See handout 
 
          Gluconeogenesis 
          PEP carboxykinase 
                    oxaloacetate ----------> PEP 
          Pyruvate carboxylase 
                    oxaloacetate ----------> pyruvate   

C₄ Plants --> Hatch and Slack pathway   

          Concentrate CO₂ via specialized cells; 
          important enzymes: PEP 
 

 

C₃ plants: many including phytoplankton 

Controls on Calvin Cycle  

RuBPcase one of 3 enzymes in cycle with "actual" (delta)G (not delta G^o´ ) far from equilibrium     -----> candidates for control points  

RuBPcase activity depends on: 
       Light; "dark" reaction, but very expensive so plants have evolved control of 

Two immediate effects on RuBPcase caused by light 
      1. pH effects: pH increases to pH=8.0 with light (protons out of stoma) 
           RuBPcase has max activity at pH 8.0 
      2. Mg²⁺ stimulates RuBPcase: Mg²⁺ go into stoma as H⁺ leave 
      3. In dark, plants synthesize analog of intermediate in CO₂ fixation reaction that binds with RuBPcase and thus inhibits it. RuBP carboxylase activase removes this analog. 

Other controls on Calvin cycle: other enzymes (e.g. FBPase) depend on redox state, i.e. whether compounds are oxidized or reduced: 

                    Light---> e^- ---> thioredoxin--->e^---> reduces FBPase, making it active 
 
 
Oceanographic applications: Correlate RuBPcase activity or amount with plant production 

Photo respiration 
      1960's noticed: plants in life can consume O₂ and evolve CO₂ 

Occurs in conditions of low CO₂ and high O₂ 
                              Rubisco 
            RuBP+ O₂ ----------> P-glycolate 
                                                         | 
                                                        \/ 
                                                  Glycolate 

                                          COO^- 
                                               | 
                                              \/ 
                                        CH₂OH 

Net result: ATP & NADPH used up. Nothing gained? 

Why photorespiration? 
      Left over from world of high CO₂, low O₂ 
      One role: protection of PS apparatus against photodamage 
            Evidence: if you blast chloroplasts without CO₂ and O₂ ---> kills it. 

      Photorespiration (PR) ---> limits plant growth (land) 
      1) Hot, bright days decrease CO₂ in chloroplasts --> P.R. can be high 
      2) Evidence that increases CO₂ due to fossil fuel burning increases plant growth