Re: How did biologists discover each step in Aerobic respiration (Formation of

Great question.  This is one of the great and complicated but under-
appreciated stories of 20th century science, even amongst professional 
biochemists.  There were a number of scientist, trained either as chemists 
or as medical doctors (biochemistry graduate training did not exist in the 
erly part of this century).  Among my favorites who contributed a great deal 
were Otto Warburg, Hans Krebs and Albert Szent-Gyorgyi.  Krebs was a post-
doctoral fellow of Warburg, and the cycle you are familiar with is named 
after him.

Otto Warburg is to my mind the father of modern biochemistry.  He is best 
known for discovering what we now call NADH and FADH2, important electron 
carriers during aerobic respiration.

These molecules can accept or give up electrons (NAD+ + 2 electrons + H+ <--
-> NADH, while FAD + 2 electrons + 2H+ <---> FADH2), and they function as 
critical links between the Krebs cycle which occurs in the cytoplasm and the 
electron transport chain which occurs in the mitochondrial membrane.  The 
electron transport chain ultimately serves to establish gradients of protons 
that whose energy is used to produce ATP, which allows many or even most of 
the reactions of life to occur.  Glycolysis, the linear sequence from 
glucose to pyruvate (which is the precursor of acetyl-CoA) that precedes 
Krebs cycle in the metabolism of glucose, also produces some NADH and ATP, 
but not nearly as much as does the Krebs cycle.  The basic steps of 
glycolysis were elucidated by about the time WWII started, and the main 
contributors were probably Warburg, Embden, Meyerhoff, Neuberg, Parnas and 
the two Coris (husband and wife).

Ultimately, the electrons that result from the oxidation of acetyl-CoA in 
the Krebs cycle, enter the electron transport chain and are finally 
transferred to molecular dioxygen (O2 - most often just called oxygen), 
which is reduced to water.  It is for this reaction that we need to breathe 
in oxygen - to serve as an ultimate electron acceptor.

As you probably know by now, the basic sequence in the oxidation of glucose 
carbon dioxide and water is: Glycolysis -> Krebs Cycle (also known as the 
Citric Acid Cycle or Tricarboxylic Acid cycle) -> Respiratory Electron 
Transport Chain and Oxidative Phosphorylation (to produce ATP).  It's all 
about electrons.  Electrons are sequentially "ripped off" of glucose during 
glycolysis and the Krebs cycle - effectively, a slow and controlled burning 
or combustion - with the end result being that glucose, a fairly reduced 
(electron rich) six carbon compound becomes the carbon dioxide - CO2, the 
most oxidized form of carbon - during the Krebs cycle.  The electrons that 
are taken from glucose during these reactions are shuttled, mainly via NADH, 
to the electron transport chain, where they "trickle" down an electron 
gradient in the (inner) mitochondrial membrane and effect the pumping of 
protons - hydrogen cations, H+ (or more exactly H3+O) - across the membrane 
(discovered later by Peter Mitchell), which allows ATP to be synthesized (a 
phenomenon known as oxidative phosphorylation).  The ultimate fate of the 
electrons is to join with oxygen to form water.  In fact, we need to breathe 
oxygen for this reason - because the respiratory electron transport chain 
needs an ultimate electron acceptor, and oxygen fits the bill excellently.

Warburg is known for this and many other discoveries and hypotheses, some 
glaringly incorrect (especially his ideas about photosynthesis).  However, a 
great deal of his impact is in establishing methods to purify and analyze 
proteins and to study biological oxidation and reduction reactions where 
none existed before.  Otto Warburg was the first biochemist to use a 
spectrophotometer, which you can use to measure the UV absorbance of a 
sample.  Many of the heme iron-containing electron transport molecules 
change their UV absorbance according to their oxidation/reduction state.  So 
this is an important measure, and was particularly important at that time 
for the study of biological oxidation and reduction.  Warburg also use an 
old tool called a manometer, which is a relatively primitive yet effective 
way of measuring gas pressure.  Clearly, changes in respiratory metabolism 
result in changes in gas pressure.  So that was also an indispensible tool 
for Warburg.

Krebs and Szent-Gyorgyi both did a huge amount of work on the Krebs cycle, 
and one could say that they really laid out the steps, even though there 
were others working on this too.  The cycle is named after Krebs, who had 
the insight to see that it was a cycle and not a non-cycling linear pathway, 
an insight that he said was due in some part to his earlier work on the urea 
cycle.

Szent-Gyorgyi is most known for his work on Vitamin C (he deduced that an 
existing sugar-like molecule was Vitamin C - ascorbic acid).  He also did 
seminal work on muscle contraction, helping to establish the roles of myosin 
and actin.  However, perhaps his least appreciated work, yet maybe his most 
important, was mapping out many of the molecules and sequence of reactions 
in what later became known as the Krebs cycle.  When Szent-Gyorgyi did his 
work, he did not know it was a cycle, but he ordered the molecules along a 
pathway that Krebs later had the insight to say turned back on itself to 
form a true cycle.

How did Szent-Gyorgyi do this?  Well, at the time, most studies of 
respiration involved using tissues that were very active in respiration, for 
obvious reasons.  A very common tissue used was pigeon heart.  Why a bird's 
heart?  Well, the heart of any animal is a very active muscle, doing a lot 
of respiration, and birds perhaps more so...they need a lot of blood pumped 
to get oxygen to their tissues during flight so that those tissues can also 
do respiration.  Why pigeons?  I don't know.  They are very common, easy to 
come by.  Maybe they were very actively bred at the time - between the world 
wars (remember, they were used as messengers in WWI).

Szent-Gyorgyi was not the first to do this (all the glycolysis people I 
mentioned earlier had used muscle extracts too in their studies), but he is 
one of the most famous to apply this technique to what we now know of as the 
Krebs cycle.  (Martins and Knoop also made some very important contributions 
in ordering the sequence of events before Krebs made it a cycle.)  He would 
grind up the pigeon hearts making heart muscle extracts and then simply add 
different chemical compounds to the extracts.  He could then measure changes 
in respiration based on changes in gas pressure.  (You can also look at ATP 
production.)  If the chemical compound could sustain respiratory metabolism, 
the reasoning was, adding a lot of it would result in large increase in the 
amount of oxygen consumed, and thus large measurable changes in gas 
pressure. This turned out to be true.  So once the basic molecules were 
found that resulted in increased metabolism when added to the ground-up 
pigeon hearts, inhibitors - mostly competitive inhibitors that were 
molecules close in structure to the active compound which blocked the 
ability of the enzymes of respiration to act - that were then being 
discovered were judiciously used by Szent-Gorgyi (and others) to deduce how 
the metabolites were ordered.  For instance, if compound A goes to compound 
B by virtue of the activity of enzyme X, blocking enzyme X with an inhibitor 
will prevent oxygen consumption from going up if you aff compound A but NOT 
if you add compound B, which comes into the pathway after the action of 
enzyme X.  So the combination of measuring changes in oxygen consumption 
with the use of chemical intermediates and inhibitors allowed these 
scientists to deduce the order of metabolites in the pathway.

Then, Krebs came along, looked at the data, did some experiments, and said 
that the pathway was actually a cycle.  It made a lot of sense and turned 
out to be correct.

As time went on, the connections between the Krebs cycle and the electron 
transport chain became clear, with the previously discovered NADH (by 
Warburg) being on of the important molecules.

Well, there's a lot more to the story and many other scientists who played 
important roles.  Many other scientists were then and after involved in 
elucidating the exact role of the mitochondria, using organelle purification 
techniques that were only devised in the late 40s, and biochemical/
biophysical techniques.  Also, the links between the different pathways 
(glycolysis and the Krebs cycle, the Krebs cycle and the electron transport 
chain and oxidative phosphorylation) were much studied later by people like 
Fritz Lipmann, Nathan Kaplan, Severo Ochoa, Feodor Lyman and many others.  
Moreover, even though it seems that other questions are more popular in 
today's biology and biochemistry, there is still a lot that is not known 
about respiration, especially the electron transport chain (since these 
proteins are embedded in the membrane, it is harder to purify and study them 
than the soluble proteins of glycolysis the Krebs cycle).  However, I hope 
that this rant give you a rough idea of how it was done.

Sincerely, Gabriel Fenteany