If the inner mitochondrial membrane is impermeable to NAD and NADH, it is somewhat of a puzzle how the NADH produced in the cytoplasm during glycolysis ever gets to the site of the respiratory chain where it can be reoxidized and its energy used to make ATP. NADH from glycolysis never enters the mitochondrion at all, but passes its free energy to a shuttle molecule that can penetrate the membrane. What is not clear is the identity of the shuttle.

In the most likely mechanism, the shuttle molecule is reduced by NADH outside the mitochondrion, diffuses inside, and then is reoxidized in the process of reducing FAD to FADH. Since the respiratory chain makes only two ATP per FADH, this represents a loss or a "toll fee" of one of the three ATP equivalents for every NADH made by glycolysis outside the mitochondrion. If this is the actual mechanism, the net production of ATP per molecule of glucose would be reduced from 38 to 36, but we will continue to use the 38 ATP figure for simplicity. This uncertainty illustrates both the shallowness of our present knowledge about some aspects of cell chemistry, and the remarkable extent to which the mitochondrion is really "outside" the rest of the cell.

Mitochondria also have their own limited genetic apparatus: DNA, polymerase and transcriptase enzymes to make more DNA and to copy the information off as messenger RNA, and ribosomes for protein synthesis. The DNA of mitochondria is small and circular, like that found in bacteria. The polymerases are different from those found in a cell nucleus, and the ribosomes resemble bacterial ribosomes rather than those of cell cytoplasm. The mitochondrion is capable of transcribing information to messenger RNA and synthesizing proteins.

A few years ago it was believed that the only proteins coded in mitochondrial DNA were some of the structural proteins of the inner membrane and cristae. Recently other proteins have been found, including some of the polypeptides of enzymes involved in the respiratory chain. However, most of these enzymes, and all other enzymes of the citric acid cycle and ATP synthesis, are synthesized from nuclear DNA in the cytoplasm, and diffuse into the mitochondria afterwards.