Explanation of mitochondrial Respiration States 1-5

           Chance and Williams  in classic J.Biol.Chem paper (Ref [1] below)  introduced the terms State 1-5 (or I-V) based on simultaneous measurements of oxygen consumption and reduced pyridine nucleotide (NADH + NADPH) fluorescence, as follows:

     When mitochondria are added to an iso-osmotic medium (IM²) containing sufficient phosphate (~1-5 mM), respiration is slow, and pyridine nucleotides in mitochondria are relatively reduced (high fluorescence). This is State 1, which represents a state of respiratory substrate starvation.

     When ADP is added, respiration increases only modestly but pyridine nucleotides become much more oxidized. Under such conditions, substrate deprivation is aggravated by high ATP demand. This is State 2.

     Then when respiratory substrate (e.g., succinate, pyruvate, etc.) is added, respiration increases markedly to a high steady rate, and pyridine nucleotides become more reduced. This is State 3.

     As State 3 continues, respiration spontaneously and markedly decreases to a slower rate, which is also nearly constant. Simultaneously, pyridine nucleotide fluorescence increases to nearly maximum. This is State 4 which occurs when all the added ADP is converted to ATP.

     Lastly, when respiration continues to the point where oxygen is exhausted and the medium becomes anoxic, oxygen uptake ceases and pyridine nucleotides become even more reduced, although not by a lot. This anoxic state is referred to as State 5.

     An uncoupler may be added to mitochondria in State 4 or in the absence of ADP(ATP), but in the presence of oxygen and respiratory substrates. This would increase the  respiration to a rate equal to that in State 3 or even somewhat higher than State 3. This is sometimes called State 3 Uncoupled, or State 3U.

     Addition of mitochondria to IM containing respiratory substrates and phosphate is accompanied by an initial short respiratory burst as mitochondria phosphorylate endogenous ADP, after which slow State 4 or "idling" or "static head" or “resting” respiration occurs. Addition of exogenous ADP then stimulates State 3 respiration which returns to State 4 when all the ADP is phosphorylated to ATP.

Comment from Dr. Starkov: modern interpretation of State 4, "resting", or "static head" respiration does not require phosphate to be present. That is, "State 4" or "resting" respiration is when  mitochondria respire slowly in the presence of substrates and oxygen. That is, no energy-dissipative duties such as cycling of an uncoupler, phosphorylation of ADP, or Ca²⁺ transport, etc.

     Thus, the famous respiratory states simply represent the consequence of sequential addition of mitochondria, ADP, and substrate to IM.

     In vivo, mitochondria operate somewhere between State 3 and State 4, sometimes called State 3 1/2 but always pronounced with tongue placed firmly in cheek.

¹The original posting by Dr. Lemasters's to the NIH MITOCHONDRIA mail list is here:

²IM may be composed of KCl, NaCl, or non-ionic osmolites such as sucrose, mannitol, et cetera. For example, most common IM compositions are: 

• Potassium based: 125 mM KCl, 2 mM KH2PO4, 1 mM MgCl2, 5 mM HEPES-KOH (pH7.2 – 7.4);
• Sodium based: 125 mM NaCl, 2 mM NaH2PO4, 1 mM MgCl2, 5 mM HEPES-NaOH (pH7.2 -7.4);
• “Cytosol” like: 110 mM KCl, 15 mM NaCl, 2 mM KH2PO4, 1 mM MgCl2, 5 mM HEPES-KOH (pH7.2-7.4);
• Non-potassium ionic: 125 mM choline chloride, 1 mM MgCl2, 2 mM H3PO4/Tris, 10 mM HEPES/Tris (pH7.2-7.4);
• Low ionic strength: 220 mM mannitol, 75 mM sucrose, 2 mM KH2PO4, 1 mM MgCl2, 5 mM HEPES-KOH (pH7.4);
• Basal osmotic minimum: 250 mM sucrose, 2 mM KH2PO4, 5 mM MOPS-KOH (pH7.4).