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 
 
CO₂ fixation: Dark reactions of photosynthesis  

          -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)  

        H₂PO + 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 

CO₂ -----> HCO₃^- CO₂ ---> Rubisco 

- keep internal CO₂ low 

- maximizes CO₂ diffusion

HCO₃^- -------> CO₂ ----> Rubisco 

+H⁺

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^o  production 11 x 10⁹ 
            Crude oil consumption 3 x 10⁹ 

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

Structure of RuBPcase 

52 kDa

13 kDa

         1. Dinoflagellates have both subunits genes in nucleus, another alga with both in chloroplast 
         2. Photosynthetic bacterium Rhodospirullum rubrum has L₂ 

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" deltaG  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 CO₂ due to fossil fuel burning increases plant growth