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Cytochrome c oxidase biogenesis: the role of the copper-binding protein CtaK

Musso, Federico (2021) MOBM02 20211
Degree Projects in Molecular Biology
Popular Abstract
Cytochrome c oxidases - why you need copper to breathe

The next time anyone will ask you to provide any proof of evolution, you can surprise them by mentioning cytochrome c oxidases. Not only this answer will make you look smart and knowledgeable, but it will also provide a very solid example of something that brings together any oxygen-breathing organism, from the smallest bacterium to a majestic blue whale. Indeed, cytochrome c oxidases are the very reason we breathe oxygen, and represent a quite irreplaceable piece of “equipment” that our cells rely on. These enzymes are in fact responsible for the crucial task of providing a final destination for the electrons that we extract from food and use as our chemical fuel to make ATP, which... (More)
Cytochrome c oxidases - why you need copper to breathe

The next time anyone will ask you to provide any proof of evolution, you can surprise them by mentioning cytochrome c oxidases. Not only this answer will make you look smart and knowledgeable, but it will also provide a very solid example of something that brings together any oxygen-breathing organism, from the smallest bacterium to a majestic blue whale. Indeed, cytochrome c oxidases are the very reason we breathe oxygen, and represent a quite irreplaceable piece of “equipment” that our cells rely on. These enzymes are in fact responsible for the crucial task of providing a final destination for the electrons that we extract from food and use as our chemical fuel to make ATP, which is in brief the main energy-carrying molecule that our cells (and not only) spend for a countless number of vital processes. As mentioned above, such electrons are combined with molecular oxygen and protons, thus generating another quite fundamental molecule for our lives: water. Whenever you’re breathing out on a cold winter day and the condensation makes your glasses foggy, that’s the product of the combined work of countless cytochrome c oxidases molecules in your mitochondria. Being electrons charged particles, however, it can be quite difficult to convince them to follow a certain path. Fortunately, millennia of evolutions provided a very fitting solution: loading our enzymes with electrically conductive metal ions. Just like the same electrons are transported to our homes along copper cables, cytochrome c oxidases have two copper and two iron clusters that direct and corral the electrons’ flow towards oxygen molecules. In my work, I focused on the first copper cluster of the cytochrome c oxidase of a very common, soil-dwelling bacterium named Bacillus subtilis. The Copper A (CuA) center, as it is called, contains two perfectly placed copper ions that act as the entry point for electrons during the last stretch of their journey. Its perfectly assembled structure, however, entails a number of challenges for its construction, as copper ions are also particularly reluctant to staying put in a specific place and can be toxic both to us and bacteria if they end up in the wrong one. This is the reason why most of our enzymes are built with the help of so-called ancillary proteins, whose only job is to help the correct assembly of other proteins. CtaK, the protein I studied, is exactly one of those. Previous studies conducted by my research group have demonstrated that CtaK is involved in the synthesis of the CuA center and that it probably binds copper ions, but the exact details had not been elucidated yet.

In order to characterize the different sections (or better, “domains”, as they are known in biology) of CtaK and to test their importance, I followed a rather common approach in molecular biology, which could be summarized as “break it down and find out”. Proteins are in fact made of several repetitive building blocks, called amino acids, which are combined in a unique way to form the protein’s three-dimensional structure. Some of them, due to their properties, are more important, and tend to be much more conserved than others during evolution. Based on this conservation, one can follow an “educated guessing” approach to identify their function. This is exactly what I did: by practicing site-directed mutagenesis on some conserved amino-acids of CtaK, I tried to find out which ones could bind copper ions and which ones are important for the protein’s stability and functioning. The experiments proceeded along two contemporary approaches, which involved testing the protein both in vivo (which means, seeing what the effect of such mutations are in the living organism) and in vitro, which implies that the protein is purified with a number of chemical steps and characterized per se. In this last process, our underlying hypothesis was that when CtaK binds copper ions, its structure is stabilized. This implies that a mutant protein which can’t bind copper ions won’t be as solid and stable as its normal, “wild-type” counterpart. The way to test this is in principle very simple: heat the protein up and see how quickly it loses its three-dimensional integrity. With the combined information from our experiments, I was able to prove that CtaK has indeed a copper-binding site in its core, in which at least three amino acids sit in a perfectly organized structure that can coordinate and transport one copper atom. Moreover, I found preliminary evidence of a potential second copper-binding site in the protein’s floppy, flexible terminal part. At the same time, my supervisor helped me to build a sketch of our long-term goal: a three-dimensional model of CtaK. Obtaining or “solving” the structure of a protein, as crystallographers say, is a much more complicated business, which implies having a very pure protein sample and convincing it to form a beautiful crystal, which is then bombarded with X-rays to see its atomic structure. This can take years! In order to have a rough idea of what CtaK could look like, we used instead a computer-based method which uses known structures of similar proteins to predict the likely appearance of CtaK.

While these findings match very well the fact that there are two copper ions in the CuA center, it’s important to remember that science requires solid proof for every argument that you bring forward! “Extraordinary claims require extraordinary evidence”, as Carl Sagan said. Our results are far from conclusive, but we have at least a starting point. Finally, in case you were wondering what the purpose of all this can be, I’ll give some examples: almost all of the modern pharmaceutical molecules are “designed” to fit and act specifically on the enzyme they’re targeting, in an overall similar process of structure determination and “rational drug design”. Moreover, as you might have read or heard, our battle against bacterial diseases is becoming harder and harder to sustain, as most of the antibiotics we developed a mere half-century ago (the blink of an eye in an evolutionary scale) are becoming increasingly ineffective. Finding out new crucial processes in bacterial biochemistry and unraveling their inner workings could be a step forward in discovering new, life-saving medicines.



Master’s degree project in Molecular Biology, 30 credits, 2021.
Biology department - Lund University

Supervisor: Claes von Wachenfeldt
Department of Biology, Lund University (Less)
Please use this url to cite or link to this publication:
author
Musso, Federico
supervisor
organization
course
MOBM02 20211
year
type
H2 - Master's Degree (Two Years)
subject
language
English
id
9067431
date added to LUP
2021-10-28 14:54:37
date last changed
2021-10-28 14:54:37
@misc{9067431,
  author       = {{Musso, Federico}},
  language     = {{eng}},
  note         = {{Student Paper}},
  title        = {{Cytochrome c oxidase biogenesis: the role of the copper-binding protein CtaK}},
  year         = {{2021}},
}