Free fatty acids cannot penetrate any biological membrane due to their negative charge. Free fatty acids must cross the cell membrane through specific transport proteins, such as the SLC27 family fatty acid transport protein. Once in the cytosol, the following processes bring fatty acids into the mitochondrial matrix so that beta-oxidation can take place.
# Long-chain-fatty-acid—CoA ligase catalyzes the reaction between a fatty acid with ATP to give a fatty acyl adenylate, plus inorganic pyrophosphate, which then reacts with free coenzyme A to give a fatty acyl-CoA ester and AMP.
# If the fatty acyl-CoA has a long chain, then the carnitine shuttle must be utilized (shown in the table below):
#* Acyl-CoA is transferred to the hydroxyl group of carnitine by carnitine palmitoyltransferase I, located on the cytosolic faces of the outer and inner mitochondrial membranes.
#* Acyl-carnitine is shuttled inside by a carnitine-acylcarnitine translocase, as a carnitine is shuttled outside.
#* Acyl-carnitine is converted back to acyl-CoA by carnitine palmitoyltransferase II, located on the interior face of the inner mitochondrial membrane. The liberated carnitine is shuttled back to the cytosol, as an acyl-carnitine is shuttled into the matrix.
# If the fatty acyl-CoA contains a short chain, these short-chain fatty acids can simply diffuse through the inner mitochondrial membrane.