Why does the reduction of NAD+ to NADH change the absorbance so much, and cause fluorescence?

Question

This is an updated question:

I don't have a very strong background in biophysics, but I want to understand the theory behind $\ce{NAD+}$ and $\ce{NADH}$ absorbance and fluorescence.

Background: I understand that the additional hydrogen that $\ce{NAD+}$ gains to form the reduced $\ce{NADH}$ results in the molecule being able to absorb light at $\pu{340nm}$. And that only the reduced $\ce{NADH}$ is able to produce fluorescence. This is also the property which is explored when studying, e.g., enzyme kinetics - if the enzyme binds $\ce{NADH}$.

Question: The oxidized form, $\ce{NAD+}$, still has an aromatic ring - why is it that it is not able to fluoresce? Why, in a nutshell, is the transfer of the hydrogens causing fluorescent in $\ce{NADH}$ and such a big change in absorbance spectrum?

Also, perhaps a bit naive question, but why is it fluorescence better to measure than absorbance?

I have tried to look for articles (the old and original) that explores these properties, but I can't seem to find any that purely speak about the spectroscopic properties.

Answer 1

Essentially NADH has an additional pair of electrons. These additional electrons which are oftein drawn as a lone pair of electrons alters the molecule in a similar way to the way in which many pH indicators change upon protonation.

Here are diagrams of the reduced and oxidized forms of NADH / NAD+

A typical indicator when protonated appears to have a colour, however upon deprotonation the indicator loses a hydrogen and gains a lone pair of electrons now appearing to have a different colour. The addition of the lone pair of electrons actually lowers the transition energy between the pi bonding and the pi anti-bonding states.

For example for methyl red it can be predicted that the unprotonated form has a HOMO/LUMO gap of 2.175 eV (according to extended Huckel calculations). For the protonated form of methyl red has a HOMO/LUMO gap of 3.275 eV.

This lowered energy correlates with a decreased absorbance energy, ultimately reflecting a higher energy wave of light (Frequency of Blue > Red). NADH's absorbance wavelength his greater than NAD+'s absorbance wavelength, because NADH is absorbing lower energy light. This, like pH indicators, is due to the lone pair lowering pi and pi anti-bonding energy state differences.