Chemiosmosis and G3P shuttle

Mitochondrial_electron_transport_chain—Etc4.svg

Chemiosmosis:

The generation of ATP by the movement of H+ across a membrane during cellular respiration. ATP synthase allows protons to pass through the membrane and uses the kinetic energy to phosphorylate ADP, making ATP.

  • The membrane must be impermeable to ions so that a proton concentration gradient can be maintained.
  • Specific transporters allow movement of ions across the membrane.
  • Electron transport through the ETC creates a proton concentration gradient in which the cystolic side of the inner mitochondria membrane has a higher conc of protons.
  • ATP synthase catalyzes ADP phosphorylation driven by the movement of protons across the inner membrane from the cytosol to the matrix.
  •  NAD and FAD are carriers pass electrons to the ETC in the inner mitochondrial membrane, which in turn pass them to other proteins in the ETC. The energy available in the electrons is used to pump protons from the matrix across the inner mitochondrial membrane, storing energy in the form of a transmembrane electrochemical gradient.
  • The protons move back across the inner membrane through the enzyme ATP synthase.
  • The flow of protons back into the matrix of the mitochondrion via ATP synthase provides enough energy for ADP to combine with inorganic phosphate to form ATP.
  • The electrons and protons at the last pump in the ETC are taken up by oxygen to form water.

 

Evidence to support postulates: 

Postulates explain the effect of uncouplers which bind to the H+ and carries them across the membrane to the matrix, thereby affecting the proton conc gradient. The second postulate demonstrates that the medium becomes acidic when mitochondria under anaerobic conditions are given small amounts of oxygen. Acidifying the media can form ATP from ADP and Pi without the need for electron transfer. ATPase activity in damaged mitochondria acts in reverse using the proton gradient to synthesize ATP i.e an ATP synthase.

G3P Shuttle:

This is an alternative mechanism that leads to the reduction of Ubiquinone.

Glycerol-3-Phosphate is converted to Dihydroxyacetone phosphate (irreversible). While this is done, mitochondrial G3P Dehydrogenase reduces E-FAD to E-FADH2 which when oxidized back to E-FAD causes Q to be reduced to QH2 which enters into oxidative phosphorylation.  When Dihyoxyacetone is reconverted to G3P (reversible), NADH + H+ is used and oxidised to NAD+.

g3p

Q Cycle and Chemiosmosis

qcyc

Q Cycle:

The modified Q cycle in Complex III results in the reduction of Cytochrome c, oxidation of ubiquinol to ubiquinone, and the transfer of four protons into the intermembrane space, per two-cycle process.

Chemical Reactions:

The first reaction of Q cycle is:
CoQH2 + cytochrome c1 (Fe3+) → CoQ- • + cytochrome c1 (Fe2+) + 2 H+ (intermembrane)

Then the second reaction of the cycle involves the reduction of the transient semiquinone by another electron to give CoQH2:
CoQH2 + CoQ- • + cytochrome c1 (Fe3+) + 2 H+ (matrix) → CoQ + CoQH2 + cytochrome c1 (Fe2+) + 2 H+ (intermembrane)

Combining the two equations, we have the overall reaction of Q cycle:
CoQH2 + 2 cytochrome c1 (Fe3+) + 2 H+ (matrix) → CoQ + 2 cytochrome c1 (Fe2+) + 4 H+ (intermembrane)

Explanation of Reactions: 

Ubiquinol (QH2) binds to the Qo site of complex III via hydrogen bonding to His182 of the Rieske iron-sulfur protein and Glu272 of Cytochrome b. Ubiquinone (Q), in turn, binds the Qi site of complex III. Ubiquinol is divergently oxidized (gives up one electron each) to the Rieske iron-sulfur ‘(FeS) protein’ and to the bL heme. This oxidation reaction produces a transient semiquinone before complete oxidation to ubiquinone, which then leaves the Qo site of complex III.
Having acquired one electron from ubiquinol, the ‘FeS protein’ is freed from its electron donor and is able to migrate to the Cytochrome c1 subunit. ‘FeS protein’ then donates its electron to Cytochrome c1, reducing its bound heme group.[1][2] The electron is from there transferred to an oxidized molecule of Cytochrome c externally bound to complex III, which then dissociates from the complex. In addition, the reoxidation of the ‘FeS protein’ releases the proton bound to His181 into the intermembrane space.
The other electron, which was transferred to the bL heme, is used to reduce the bH heme, which in turn transfers the electron to the ubiquinone bound at the Qi site. The movement of this electron is energetically unfavourable, as the electron is moving towards the negatively charged side of the membrane. This is offset by a favourable change in EM from -100 mV in BL to +50mV in the BH heme. The attached ubiquinone is thus reduced to a semiquinone radical. The proton taken up by Glu272 is subsequently transferred to a hydrogen-bonded water chain as Glu272 rotates 170° to hydrogen bond a water molecule, in turn hydrogen-bonded to a propionate of the bL heme.
Because the last step leaves an unstable semiquinone at the Qi site, the reaction is not yet fully completed. A second Q cycle is necessary, with the second electron transfer from cytochrome bH reducing the semiquinone to ubiquinol. The ultimate products of the Q cycle are four protons entering the intermembrane space, two protons taken up from the matrix and the reduction of two molecules of cytochrome c. The reduced cytochrome c is eventually reoxidized by complex IV. The process is cyclic as the ubiquinone created at the Qi site can be reused by binding to the Qo site of complex III.

http://www.youtube.com/watch?v=CKBeQYr66TQ

http://www.youtube.com/watch?v=t4jeOEerOD8

Electron Transport Chain

ETC couples electron transfer between an electron donor (such as NADH) and an electron acceptor (such as O2) with the transfer of H+ ions  across a membrane. Protons are pumped from the mitochondrial matrix into the intermembrane space creating an  electrochemical proton gradient which allows ATP synthase (ATP-ase) to use the flow of H+ through the enzyme back into the matrix to generate ATP from ADP and inorganic phosphate. The mitochondria consists of a double membrane, the outer is permeable to most small molecules and ions while the inner is impermeable to most small molecules hence specific transporters are required. ETC takes place in the inner membrane through 4 complexes. There are 5 types of electron-carrying molecules that shuttles electrons along these complexes; NAD+, FAD/FMN, Qbiquinone, Cytochromes and Iron-Sulphur Proteins.

Summary through ETC: Complex I accepts electrons from the Krebs cycle NADH, and passes them to coenzyme Q, which also receives electrons from complex II, UQ passes electrons to complex III, which passes them to cytochrome c. Cyt c passes electrons to Complex, which uses the electrons and hydrogen ions to reduce molecular oxygen to water.

Complex I:

In Complex I (NADH dehydrogenase) two electrons are removed from NADH to FMN to form FMNH2.  Electrons are then passed one at a time (through a series of Fe-S clusters) to lipid-soluble carrier, ubiquinone (Q) to form semiquinone intermediate then QH2. The reduced product, ubiquinol (QH2) freely diffuses within the membrane, and Complex I translocates four protons (H+) across the membrane, thus producing a proton gradient.
The pathway of electrons occurs as follows:

NADH + Q + 5H+ (matrix) –> NAD+ + QH2 + 4H+

  • NADH is oxidized to NAD+, by reducing Flavin mononucleotide to FMNH2 in one two-electron step.
  • FMNH2 is then oxidized in two one-electron steps, through a semiquinone intermediate.
  • Each electron thus transfers from the FMNH2 to an Fe-S cluster, from the Fe-S cluster to ubiquinone (Q).
  • Transfer of the first electron results in the free-radical (semiquinone) form of Q, and transfer of the second electron reduces the semiquinone form to the ubiquinol form, QH2.
  • During this process, four protons are translocated from the mitochondrial matrix to the intermembrane space.

Complex II:

In Complex II (succinate dehydrogenase) additional electrons are delivered one at a time through three Fe-S centres to Q to form QH2. Complex II consists of five prosthetic groups and four protein subunits: SDHA, SDHB, SDHC, and SDHD. C and D are integral membrane proteins with three transmembrane helices. Other electron donors (e.g., fatty acids an glycerol 3-phosphate) also direct electrons into Q (via FAD). Complex II is NOT a proton pump. b Heme does NOT play a direct role in electron transfer.

Complex III:

Structure: The enzyme is a homodimer with 11 distinct polypeptide chains. Major prosthetic groups; 3 hemes and a Riske 2Fe-2S cluster which mediate electron-transfer between Q in the membrane and Cytochrome C in the intermembrane space.

Redox Chemistry:

  • In Complex III (cytochrome bc1 complex), the Q-cycle contributes to the proton gradient by an asymmetric absorption/release of protons.
  • Two electrons are removed from QH2 at the QO site and sequentially transferred to two molecules of cytochrome c.
  • The two other electrons sequentially pass across the protein to the Qi site where Q is reduced to QH2.
  • A proton gradient is formed by two quinol (4H+4e-) oxidations at the Qo site to form one quinol (2H+2e-) at the Qi site.
  • In total six protons are translocated: two protons reduce quinone to quinol and four protons are released from two ubiquinol molecules.

Complex IV:

Structure: Dimer formed by a protein monomer composed of 13protein subunits. Three subunits form the central core of the enzyme. Major prosthetic groups includes CuA/CuA, Heme A and Heme a3-CuB. Heme a3-CuB is the site of reduction of O2 to H2O.

Redox Chemistry: In Complex IV (cytochrome c oxidase), sometimes called cytochrome A3, four electrons are removed from four molecules of cytochrome c and transferred to molecular oxygen (O2), producing two molecules of water. At the same time, four protons are removed from the mitochondrial matrix (although only two are translocated across the membrane), contributing to the proton gradient. The activity of cytochrome c oxidase is inhibited by cyanide.

Cytochrome C Oxidase Mechanism:

  • Two molecules of Cytochrome C used to sequentially transfer electrons to reduce CuB and Hemea3
  • Reduced CuB and Fe in Hemea3 bind O2, which forms a peroxide bridge.
  • Addition of two more electrons and two more protons cleave the peroxide bridge.
  • Addition of two more protons lead to the release of water.

Peroxide bridge: Oxygen bound to hemea3 is reduced to peroxide by the presence of CuB.

Total protons moved per 2e:

4H+ pumped at complex I. 2H+ taken from matrix, 4H+ released at the IMS at complex III. 2H+ pumped and 4H+ taken up from the matrix at complex IV. For NADH 10 protons pumped and for FADH2 6 protons pumped.

Inhibitors:

  • Complex I: Rotenone and Piericidin A
  • Complex II: Malonate
  • Complex III: Antimycin A (Qn) , Myxothiazol (Qp) and Stigmatellin (RFe-S)
  • Complex IV: Cyanide, CO

 

TCA cycle

After glycolysis has occurred  once oxygen is available, Acetyl-CoA is produced and it enters the Krb’s or TCA cycle which takes place in the matrix of the mitochondria. TCA cycle is easily explained in the form of a diagram:

tca themedicalbiochemistrypage.org

 

The following videos are also very useful!

http://www.youtube.com/watch?v=9EFRHRXPDWo

http://www.youtube.com/watch?v=juM2ROSLWfw

http://www.youtube.com/watch?v=WXHpTHb1MQM

Kreb’s/ TCA Cycle Video Review

As i previously mentioned, I usually watch a few youtube channels when studying, as it often makes learning fun and easy. Another one of my favourite channels is ThinkWell, who also has a website with a lot of great information on biology and chemistry topics. I came across one of his videos on TCA and will now do a review on it:

http://www.youtube.com/watch?v=9EFRHRXPDWo

Summary of the video:

The video basically breaks down TCA step by step, showing and explaining the mechanisms of each reaction, and ending with the first reactant being the last product, hence emphasizing the cyclic part of the Kreb’s Cycle.

  • His animated voice as well as excessive hand gestures kept the video interesting as well as full attention throughout the entire video.
  • Every single reaction of TCA is explained individually and in depth with mentions including but not limited to: reactions, reactants,  chemical structure of products and reactants, enzymes and by-products.
  • Cutting of compounds showed the physical breaking and forming of bonds example S-CoA.
  • The type of each reaction occurring is mentioned which included but is not limited to: oxidation, NAD reduction, destabilisation, exothermic reactions and subtrate-level phosphorylation.
  • The way in which each reaction occurs and products formed is demonstrated.
  • A downloadable paper to follow along with the video is available on the channel’s offical website: http://www.thinkwell.com

However, one way in which the video can be improved is if a white board is used instead of so many paper so that paper can be saved.

In conclusion, i believe that the video is amazing, makes learning fun and easy while at the same time covering the necessary information.

goodbye (1) www.tangrammedia.com

 

Enzyme video reveiwed

While cramming for subjects, i usually use a couple youtube channels that has concise information about topics. One of my favourite channel is BrightStorm2 which has a range of topics in biology,chemistry,physics and math. The following is a video from their channel on Enzymes:

http://www.youtube.com/watch?v=870MWm0peRI

Summary of the video:

The lecturer talked about enzymes being a protein catalyst, having a substrate specific active site, induced fit theory, and the effect of pH and temperature.

  • Firstly, i thought that the video was concise, but filled with enough information to give you a general idea of Enzymes.
  • His animated voice kept your attention throughout the entire video.
  • The use of a diagram of an Energy vs Time graph on the white board gave you a visual understanding of how enzymes work as a catalyst and lowers activation energy and speeds up the rate of reaction.
  • The use of main points on the white board also gave a visual of the main characteristic of Enzymes that should be known. these main points were also discussed in the video: Protein catalyst, active site, specific to substrate, induced fit theory, pH and temperature.
  • The use of the scissors gave a real life application and therefore better understanding of enzymes being a catalyst and the active site being substrate specific.
  • The use of assistant Laura also gave the video an added touch of familiarity and proved that enzymes act as catalyst and make reactions easier to occur.
  • Diagram of induced fit theory gave a visual, hence better understanding of how the theory works.

 

However, the video can be improved in multiple ways:

  • The video could have been edited so that Laura ripped the paper correctly the first time, proving that enzymes make reactions occur easier.
  • Other topics such as Inhibition, Nomenclature, Enzyme classes, Inorganic catalyst vs biological catalyst, M-M equation, Line-weaver Burk plot and Allosteric enzymes could have been covered. This could have given the video a sense of depth instead of a general idea. In this way, students at university level would find the video very useful.
  • Other graphs such as pH and temperature graphs could have been used.
  • pH and temperature effect on enzymes could have been explained more, instead of simply mentioned.

 

In conclusion, i believe the video is best for cramming purposes, because it give a general sense of Enzymes and their characteristics.

goodbye www.maurilioamorim.com

Is Methylene Blue a cure for a disease? [Published paper #2]

Source: http://www.scientificamerican.com/article.cfm?id=common-lab-dye-found-to-i

So there I was browsing through scientific websites looking for a topic to do my assignment on, and I came across a chemical that I’ve used in biology and chemistry labs before, and apparently it has properties that help in Huntington’s Disease.  This is being researched by Leslie Thompson, a neurobiologist at University of California–Irvine and her team. Leslie Thompson is in the picture below:

Imagehttp://faculty.uci.edu/profile.cfm?faculty_id=4708

Huntington’s disease (HD) is a genetic disorder that disturbs muscle coordination and results in mental deterioration and psychiatric issues. It is usually recognizable in adults in their 30s and 40s. HD is the most common genetic cause of abnormal involuntary writhing movements called chorea. Huntington’s disease occurs when the C-A-G sequence of DNA base pairs repeats too often on the HTT gene, creating a long version of the Huntington protein, which therefore folds incorrectly and produces clumps in the brain. HTT is a protein that interacts with many other proteins as well as has many biological functions. HD is not caused by inadequate production of HTT, but by an accumulation of the toxic function mHTT. It is a neurodegenerative disease, which causes a gradual loss of structure or function or death of neurons.

Imagefolding.stanford.edu

Below is a microscope image of a neuron with inclusion (stained orange) caused by HD, image width 250 µm:

Imagehttp://en.wikipedia.org/wiki/Huntington’s_disease

Methylene blue is said to disrupt the formation of mHTT protein clumps in HD. Methylene blue was used to treat ailments from cyanide poisoning to malaria from since 1897. However, Food and Drug administration has never officially acknowledged it as a therapy for any diseases or illness. There is currently no drug produced to stop HD progression. Methylene blue itself is not harmful to humans.

The research team is currently experimenting with flies and mice. Drosophila flies with mHtt gene were given food mixed with methylene blue for seven days. Results of the flies’ brains showed that protein clumps had been reduced by 87 percent compared with a control group. The mice with the mHTT gene were tested for mobility. The 2-month old treated mice demonstrated irregular clasping of their hind claws only 20 percent of the time in a reflex test, while the untreated mice clasped at a 60 percent rate. Less clasping meant healthier mice. However, the amount of mice used was not large enough to give statistically feasible results and the difference in the tests “dropped off” after 9 weeks.

Imagehight3ch.com

The research team says a lot more research on methylene blue is needed but they are hopeful because the early steps of clumping of the mHTT protein is significantly altered in test tubes, the flies and also the mice. They state that methylene blue may prevent mHTT from sticking to itself. Thompson highlights that “Methylene blue would absolutely require further testing in mouse models and would need safety and efficacy trial before it could be used for humans.”

Now think about the difference that a chemical we use as a stain and indicator..has the ability to save the millions of people that suffer from HD worldwide. Furthermore, what other chemicals purposes are being underrated and not utilized? I really hope that the clinical trials go well and methylene blue is approved as a therapy for Huntington’s Disease.

Imageblogs.nature.com

On curing HIV..[Published Paper #1]

Published Paper Source: http://www.scientificamerican.com/article.cfm?id=new-protein-blocks-hiv

A team of researchers (led by Peter S. Kim) of the Whitehead Institute for Biomedical Research has discovered a protein that can block HIV.

Image

The protein is 5-Helix and it blocks HIV entry into the body when it binds to a region of the HIV protein coat known as gp41 and therefore act as an entry inhibitor. To understand what this means, you must first understand how HIV enters into a human cell. This is done in 5 steps:

  • Binding of HIV surface protein gp120 to CD4 receptor.
  • A conformational change in gp120 increases its affinity for a co-receptor and exposes gp41
  • Binding of gp120 to co-receptor CCR5 or CXCR4
  • Penetration of the cell membrane by gp41. This approximates the membrane of HIV and T cell and promotes their fusion.
  • Entry of the viral core into the cell.

Image

Protein 5-Helix therefore prevents step 4, and gp41 does not penetrate the cell membrane when they bind together. Step 5 is avoided by consequence and the viral core is prevented from entering into the human cell.

HIV is always mutating, it never becomes fixed and 5-Helix also seems to successfully prevent a wide range of HIV strains and hence would be useful in creating a new class of anti-HIV drugs. 5-Helix may even help in fighting other viruses like Ebola, human respiratory syncytial virus (HRSV) and influenza all of which show similar characteristics to HIV.

On the other hand, 5-Helix can be used as preventative treatment or a vaccine.

T-20 is another entry inhibitor that also may have had the same effect as 5-Helix but it was required in large amounts to give a positive result while 5-helix gave the positive result in little amounts.

5-helix inhibits HIV in cell culture.The research team is now determining if 5-helix works in animal models so that they can develop it for humans.

I found this article to be particularly interesting because of the recent developments in HIV which includes a baby and 14 people that were ‘functionally cured’ of HIV. This story can be found in the following article:

http://www.newscientist.com/article/dn23276-more-hiv-cured-first-a-baby-now-14-adults.html

References for pictures:

www.avert.org

blogs.delawareonline.com

Glycolysis Quiz

Its quiz time once again..so put on your thinking caps and make me proud biochemists!

blog26

1) What is the most regulated enzyme?

a)      Adolase

b)      Phosphofructokinase-1

c)       Phosphohexoseisomerase

d)      Enolase

e)      Pyruvate Kinase

2) What co-factor is needed for the conversion of Pyruvate to Acetyl-CoA?

a)      Mg2+

b)      TPP

c)       Cu2+

d)      Vitamin C

e)      All the above

3) What is the co-factor that all kinase enzyme require?

a)      Fe2+

b)      Cu2+

c)       Vitamin A

d)      TPP

e)      Mg2+

4) Which reaction is the only oxidation phase in glycolysis?

a)      Fructose-6-phosphate to Fructose-1,6-bisphosphate

b)      1,3-BPG to 3-Phosphoglycerate

c)       G3P to 1,3-BPG

d)      Phosphoenolpyruvate to Pyruvate

e)      Glucose to Glucose-6-Phosphate

5) In which reactions are ATP produced?

a)      1st and 10th reactions

b)      All reactions of the Energy Generation phase

c)       7th and 10th reactions

d)      Reactions vary according to cell

e)      ATP is produced in the Citric cycle and consumed in Glycolysis

6) In which reactions are ATP consumed?

a)      1st and 3rd reactions

b)      All reactions of the Energy Investment Phase

c)       2nd and 5th reactions

d)      ATP is only produced not consumed

e)      Glucose to Glucose-6-phosphate ONLY

7) Where does glycolysis occur in the cell?

a)      Cytosol

b)      Cytoplasm

c)       Mitochondria

d)      Nucleus

e)      Directly outside the cell

8) Multiple Answer MCQ:

Select the correct multiple choice answer using ONE of the keys A,B,C,D or E below:

a)      1 and 4 are correct

b)      2 and 3 are correct

c)       Only 4 is correct

d)      1 and 3 and 4 are correct

e)      2 and 4 are correct

8) Which reactions are irreversible?

1)      Glucose-6-Phosphate to Fructose-6-Phosphate

2)      Glucose to Glucose-6-phosphate

3)      DHAP to G3P

4)      Phosphoenolpyruvate to Pyruvate

9) Why is/are this/these reaction(s) irreversible?

a)      Backward reaction is not energetically feasible

b)      Not enough product is made for a reverse reaction

c)       Backward reaction produces too much heat

d)      Change in structure of product prevents reactants from being made.

e)      All of the above

10) What enzyme is used to convert Glucose-6-Phosphate to Fructose-6-Phosphate?

a)      Aldolase

b)      Enolase

c)       Phosphohexose isomerase

d)      Phosphofructokinase-1

e)      Glucose-6-Phosphase

11) What is the by-product when Enolase converts Phosphoglycerate to Phosphoenolpyruvate?

a)      NADH

b)      H2O

c)       ATP

d)      DHAP

e)      TPP

12) What happens to pyruvate when oxygen is unavailable?

a)      It is converted to L-Lactate

b)      It is converted to Ethanol and enters Citric cycle

c)       It is converted to Acetaldehyde and enters Citric cycle

d)      It is converted to Acetyl-CoA and enters Citric cycle

e)      All the reactions of Glycolysis are reversed