Good day ladies and gentlemen,
Since chlorophyll exposed to light but isolated from the cell is photochemically inactive,
the process of photosynthesis is dependent upon enzymes, as respiration and fermentation are.
In the following, I would like to acquaint you with some aspects of enzymes involved in photosynthesis.
The enzymes of photosynthesis are not understood nearly as well as those involved in respiration and fermentation,
but you will see nevertheless that we can say quite a bit about them today.
The first slide, please. We shall first examine a process that is the opposite of photosynthesis, a process of respiration,
indeed the oxidation of glucose phosphate to pentose phosphate by means of nicotinamide in the equation written here.
The reaction was discovered in Berlin-Dahlem shortly after the discovery of the pyridine nucleotide.
It takes place in two steps. Firstly, one molecule of nicotinamide oxidises the glucose phosphate to glucose phosphoric acid,
then an additional molecule of nicotinamide oxidises the hexonic phosphoric acid to pentose phosphate
by splitting off carbonic acid. A specific enzyme is required for both of these steps.
In the reaction written down here, the glucose phosphate is therefore oxidised to pentose phosphate
by two enzymes whose active group is nicotinamide and that transfer hydrogens.
One would think that the complete combustion of glucose then continues similarly,
so that pentose phosphate is oxidised to tetrose phosphate, the tetrose phosphate to triose phosphate, and so on.
Horecker says, however, that this is not the case and instead that pentose phosphate is divided into smaller fragments
by a cleaving enzyme after it has been formed by the oxidation reaction written here,
and that the fragments are then condensed again into glucose phosphate,
so that the oxidation always only takes place with formations of six carbons.
If glucose phosphate has been oxidised six times to pentose phosphate,
then the net result is that one molecule of glucose phosphate has been burned.
Through this sensible mechanism,
organisms manage to complete the oxidation of the six carbon atoms in glucose with two enzymes,
while without the regeneration of glucose, six times two = twelve oxidation enzymes would have been necessary.
It can also be said of this mechanism that the pentose phosphate formed through oxidation is not arabinose phosphate
as one should have expected, but instead is ribulose phosphate, the phosphate of a ketopentose,
one which has been very rarely found in nature until now.
It can moreover be said that a further, very rare sugar appears as an intermediate product
during the recombination of the pentose fragments into the glucose phosphate: the phosphate of sedoheptulose,
a ketose with a seven-carbon chain.
These rare sugars, ribulose and sedoheptulose, are thus intermediate products of burning glucose through nicotinamide.
Now you will ask what these very specialised facts about glucose oxidation have to do at all with photosynthesis.
Actually, quite a bit. Because during photosynthesis with radioactive carbonic acid at Calvin's institute in Berkeley,
carbon bonds were found sixty seconds after applying light that are none other than the phosphates of ribulose and sedoheptulose.
I should add that the enzyme reactions written here also run from right to left in vitro using pure enzymes,
if one starts with pentose phosphate and dihydronicotinamide,
so there can be no doubt that the reduction of the carbonic acid during photosynthesis
is caused by the reverse reaction of the respiration processes written here.
Hydrogenated nicotinamide occurs during respiration and is always reoxidised again by the molecular oxygen.
Photohydrogenated nicotinamide occurs during photosynthesis and is always reduced again
by chlorophyll that has been exposed to light.
It is therefore a reduction of carbonic acid by photosynthesis, not just as a net outcome,
but rather within the mechanism as well, the reverse reaction of a respiration process, a known respiration process.
The identical hydrogen-transfer enzymes are respiration enzymes or photosynthesis enzymes,
depending on whether they are supplied with nicotinamide or hydrogenated nicotinamide.
It may be that the carbonic acid is reduced during photosynthesis not only through the reaction described here,
but also through other reactions.
For example, the reverse reaction of decarboxylising malic acid to pyruvic acid, which Ochoa in New York in particular believes.
But the principle is always the same. Everyone agrees that it is always the reductive carboxylation of hydrogenated nicotinamide
by which the carbonic acid is bound and reduced during photosynthesis. Lights, please.
While on one hand the reduction of carbonic acid in photosynthesis is the reverse reaction of a respiration process,
the forward reactions of respiration also play an important role in photosynthesis.
In the primary reaction of photosynthesis, it is well known that one mole of carbonic acid is absorbed
and one mole of oxygen generated when one mole of light quanta are absorbed.
But since 112,000 calories are necessary to generate one mole of oxygen from carbonic acid,
while the energy from one mole of photos in the red wavelength only amounts to 42,000 calories,
nature decided as an alternative that the energy lacking from light quanta,
namely the significant amount of two-thirds of the total energy,
will be made available to photosynthesis from the chemical energy stored in the cells.
That is one solution to the so-called quantum problem in photosynthesis,
which many researchers in the natural sciences have worked on since about, that has been going since 1920,
which is thirty-five years. None of us had remotely thought about this mechanism, about this alternative.
We always wondered how it is possible that three quanta, which are necessary, how they can collaborate,
when in reality it is accomplished simply by the cell adding as much chemical energy as the light quanta lack.
That is an almost unbelievably simple and brilliant mechanism.
And you actually see in this example that organisms are indescribably cleverer than researchers in the natural sciences.
Because for thirty years, this idea didn't occur to anyone in the slightest until it was actually observed on a manometer.
It was seen on a manometer. When light was applied, there was a pressure of one quantum and as soon as it was dark,
two-thirds disappeared again and everything was then thermodynamically balanced.
Now the regeneration or the entire mechanism would make no sense of course, if the cells were to add energy and thereby lose it.
But as mentioned, it so happens that the cell immediately receives the energy back
and indeed receives it through a respiration process
in which the cell always receives energy back via the light of photosynthesis, which has quasi been loaned out.
Photosynthesis, as it can also be described, is therefore only possible
through a continuous consumption and regeneration of chemical energy in the cell,
without which the light entering the green cells has no effect.
So first respiration - it can also be expressed this way - creates the photosensitivity of the green cells.
And that is why the enzymes of respiration also are part of the enzyme chain of photosynthesis.
The experiments that prove this have been developed in Dahlem in such a way that they are beyond any doubt.
You can imagine that when something like this is discovered, no one believes it at first.
It is always like this, as soon as one encounters something new, there is immediate opposition, which is understandable and human.
It was thus suggested that the effects are too small, that it only comes from heat.
In reality, the situation now is that we get thirty-five cubic mm of oxygen in one minute when we illuminate a sample.
Now, if you calculate that the error is perhaps 0.5 cubic mm, then the matter is beyond all experimental doubt.
Then you proceed to illuminate the sample so strongly that all of the available cell energy is consumed.
Thus you can virtually titrate out the cellular existing energy by illuminating so strongly that all of it is extracted.
When that has happened and it is illuminated again, you no longer get a quantum yield of one
because of course the cellular energy is now depleted. And then you have to wait.
You have to wait approximately ten minutes in the dark, and if you then illuminate the sample again, you get energy again.
But if you illuminate the sample during the intervening period, you get less.
After two minutes you get perhaps six cubic mm, after ten minutes you get twelve, and so on.
So it is evident that the cellular energy is actually recovered during the recuperative process of oxygen consumption.
To prove that it is oxygen you can proceed as follows:
you illuminate a sample strongly and extract all of the cellular energy that is there.
Then you take away the oxygen from the cell.
Now you can wait as long as you wish and when you illuminate the sample again,
you never get the oxygen production in light again, because the cell does not get its energy back.
Similarly, when you introduce oxygen again and illuminate the sample, you get oxygen production again with a quantum yield of one.
You can therefore really prove and measure, track how the cell gets its energy back
and that in fact without this cellular energy, the entire system of the green cells does not react to light at all.
That the photosensitivity of the green cells is actually created initially by respiration
makes photosynthesis different from all other photochemical reactions of the inanimate world.
As I began with, that chlorophyll isolated from the cell is photochemically inactive,
we can now add that chlorophyll found within the living green cell is also photochemically inactive
if one single substance among all of the substances of the cell is missing,
namely the substance that is the vehicle of the auxiliary chemical energy.
Its chemical nature remains unknown.
The only thing certain is that it is not adenosine triphosphate, as has sometimes been assumed in chemical literature of late.
Because the amounts of maximum energy that you can titrate out of the cell
and that can be made available by the cell are so large that if you were to identify and assume it was phosphorus,
that all of the phosphorus is bound in adenosine triphosphate,
then you would still not have explained even one-third of the auxiliary energy that is actually made available,
measured by the oxygen coming from the cell.
So, first we have had the bonding, the reduction of carbonic acid as a reverse reaction of a respiration process,
then here we have the forward reaction of respiration, without which photosynthesis is impossible.
That makes two occurrences of respiration enzymes, simultaneously photosynthesis enzymes.
A third matter, which is only - that I still will report on briefly to you now - only exists in photosynthesis.
I must first make a remark about the pigments of the green cells and their photoabsorption.
All green cells contain, besides the chlorophyll, yellow pigments, the carotenoids.
While chlorophyll absorbs over the entire visible spectrum,
the yellow pigments absorb only in the short-wavelength portion of the visible spectrum,
and indeed most strongly in blue-green, that is near the blue-green cadmium lines.
If you illuminate green cells with blue-green,
approximately half the light will be absorbed by the chlorophyll and the other half of the light by the yellow pigments.
While in red, only the chlorophyll absorbs and the yellow pigments not at all.
Depending therefore on whether you shine red or blue-green light on the green cells,
the yellow pigments can share in up to fifty percent of the absorption or not at all.
Our experiments associated with these effects assumed then that the yield of chemical energy in photosynthesis by Chlorellas
was generally better in summer than in winter.
Now our Chlorellas are always raised under incandescent light, in summer as well as winter,
but in a south-facing window so that the cells also have always received daylight besides the incandescent light when grown.
Daylight is known in medium-short wavelengths like incandescent light,
and contains much more blue-green light than incandescent light.
We therefore wondered whether it would not perhaps be appropriate during measurements of the photosynthetic yields
to augment light in the red region or the monochromatic green with short-wavelength light
and so possibly improve the yields of the winter cells.
In fact we found that the yields rose when we augmented red or green monochromatic light with shorter wavelength light.
The most effective was blue-green. Out of these observations - please, next, next slide -
grew the present-day experimental procedure for measuring the yield of photosynthesis.
In this experiment, for example, which is a typical experiment with winter cells,
Chlorellas are continuously irradiated with very high intensity red light.
This experiment lasts about seven hours for example.
Then light, which is blue-green in colour, alternately augments it and the experiment is continued again.
Now this is very important for the experimental procedure, because it greatly simplifies the interpretation:
the blue-green light that acts is energetically almost equal to zero compared to the red light, whose energy turnover you measure.
The blue-green light that you apply is active yet cannot participate in the oxygen production,
but instead only can act catalytically.
Now, if you look at the illustration, there is one more thing to say: In general,
you express the quantitative results of photosynthesis measurements using the so-called quantum requirement
that is 1 divided by phi and means the mole of quanta absorbed divided by the mole of oxygen produced.
The quantum requirement thus becomes larger, the worse the yield is.
I want to address you one more time about this, because this experiment, how it was run,
and want to tell you about the energy yield for it, converted.
After two hours, you have to imagine, the cells come out of the dark, after two hours of blue-green,
the quantum requirement is 4.4, which corresponds to a 60% chemical energy yield.
Then the blue-green exposure is taken away.
And almost immediately, the quantum requirement rises to infinity.
That means there is absolutely no oxygen produced any longer and the chemical energy gain is zero.
Then we apply blue-green light again.
The quantum requirement falls to 3.6, which means that the yield of chemical energy climbs to about 75%.
We then take the blue-green away again and the quantum requirement rises to 17,
which means that the yield of chemical energy falls to 16%.
We then add the blue-green again and the quantum requirement falls to 3.2, that means the yield of chemical energy rises to 83%.
The blue-green is taken away again and the quantum requirement rises immediately to 12,
that means the yield of chemical energy falls to 22%.
All together, the yield of chemical energy in the experiment in the absence of blue-green light has therefore been very poor
and in the presence of blue-green very good.
If you wanted to, which we have not done, add in the respired oxygen to the oxygen produced by the light,
the quantum requirement in the last two blue-green periods would be 2.85 on average,
which corresponds to a 93% chemical energy yield.
Now, the explanation. Blue-green light, as already mentioned, is strongly absorbed by the carotenoids.
We also know that the carotenoids sustain a cis-trans rearrangement through illumination.
We also know about them that the cis-trans rearrangements are exploited by organisms,
rhodopsin is in fact a carotenoid according to the investigations by George Wald,
and the effect of light upon rhodopsin is based on the cis-trans rearrangements of the visual carotenoids -
or the visual carotenoid.
Since, from the experiments written down here, a small amount of blue-green light creates large energy turnovers in Chlorellas,
it can be concluded that a carotenoid isomer that forms in light is the active group of an energy conversion enzyme.
This carotenoid isomer may be more stable in summer cells than in winter cells.
And so it might be that the influence of the blue-green light in experiments has been discovered with winter cells.
We always add blue-green light now and almost always find a significant improvement
from the blue-green in the summer cells as well, but not as strong as in winter.
I believe that this discovery will acquire a similar significance for the chemistry of photosynthetic enzymes
as did the discovery of carbon monoxide and effects of light for the chemistry of respiration enzymes years ago.
In any case, blue-green light is already beginning to explain the hitherto-unknown function of carotenoids in photosynthesis.
To summarise, today we are able to write down the equation of the enzyme reaction
by which carbonic acid is reduced in photosynthesis.
- please, the third slide - But we cannot yet write down the equation for the conversion of light energy to chemical energy.
Equations such as these - and the fourth slide, once again the third.
So you see here two equations that contain the following facts: firstly, the nicotinamide, dihydronicotinamide,
as a catalyst for the reduction of carbonic acid; secondly,
dihydrocarotene and carotene as accounting for the experiments with blue-green light.
Thus, both of these facts are accommodated in these equations.
But these equations are surely wrong, because they would be ignoring the most important result
of all the photosynthesis experiments, the quantum yield of 1 for the primary reaction of photosynthesis.
In this equation here you have 4, which cannot therefore be correct.
In the equations of photosynthesis, the energy conversion of photosynthesis, the unknown substance belongs,
that initially facilitates the activity of the low-energy quanta of visible light in the cycle of breakdown and regeneration.
And with that, I have discussed roughly everything that we can say about the enzymes of photosynthesis at present.