What is the pH of a cell? Hydrogen ions play a central role in the lives of cells. For example, as illustrated by the wide variety of pK values for different macromolecules and specifically for proteins, changes in hydrogen ion concentration are intimately tied to the charge of side chains in proteins. This charge state, in turn, affects the activity of enzymes as well as their folding and even localization. Further, certain key molecular machines of the cell such as the famed ATP synthases that churn out the ATPs that power many cellular processes are driven by gradients in hydrogen ions across the membranes that occupy them. The abundance of these ions and, as a result, the charge state of many compounds is encapsulated in the pH defined as pH log 10 [ H ] [1M ]) where [] denotes the concentration or more formally the activity of the charged hydrogen ions (H+ or more accurately the sum of hydronium, H3O+, as well as the functionally important but often overlooked Zundel, H5O2+, and Eigen, H7O3+, cations). We are careful to divide the hydrogen ion concentration by the so-called “standard state” concentration, namely 1M, in order to ensure that when taking the log we have a unitless quantity. This step is often skipped in textbooks. The integer 7 is often etched in our memory from school as the pH of water, but there is nothing special about the integral value of 7. Water has a neutral pH of about 7, with the exact value varying with temperature, ionic strength and pressure. What is the pH inside the cell? Just like with other parameters describing the “state” of molecules and cells, the answer depends on physiological conditions and which compartment within the cell we are considering (i.e. which organelle). Despite these provisos, crude generalizations about the pH can be a useful guide to our thinking. An E. coli cell has a cytoplasmic pH of ≈7.2-7.8 (BNID 106518). This pH value is a result of both active and passive mechanisms required for the ability of E. coli to colonize the human gastrointestinal tract with niches of pH ranging from 4.5 to 9 (BNID 106518). A passive mechanism for maintaining this pH relies on the buffering capacity provided by the cell’s metabolite pool, that is, the fact that a change in pH will result in a release or absorption of hydronium ions which counteracts the original change. As shown in the vignette on metabolite concentrations within cells, the main ingredients of the metabolite pool are glutamate, glutathione and free phosphate, all with concentrations in the mM range, i.e. millions of copies per bacterial volume and a pKa in the neutral range ensuring that they will tend to counteract changes in pH. Active mechanisms for controlling the hydrogen ion concentration include the use of transporters such as ATPases, that are driven by ATP hydrolysis to pump protons against their concentration gradient. These transporters are regulated such that the cell can actively involve them in order to sculpt the intracellular pH. As a second depiction of an organism’s characteristic pH range, budding yeast is reported to have a cytoplasmic pH of ≈7 when growing on glucose (Orij 2009, but also see Valli, 2005 for lower values). As shown in Figure 1A, these measurements were carried out using more fluorescent protein tricks, this time with a pH-sensitive fluorescent protein. By examining the ratio of the light intensity emitted by these proteins at two distinct wavelengths, it is possible to calibrate the pH as shown in Figure 1B. Yeast flourish when the external pH is mildly acidic as their transport into the cell is often based on co-transport with an incoming proton and is thus more favorable if the external pH is lower than its value within the cell (Orij 2011). Pumping excess protons to the vacuole is an efficient way of maintaining a high cytoplasmic pH, indeed the vacuole has a more acidic pH of ≈5.5-6.5, while these same fluorescence measurements reveal that the yeast mitochondria are characterized by a pH of 7.5. Interestingly, as shown in Figure 1C, the internal pH of these cells is kept almost constant under very different pH of the surrounding medium. Using such pH sensitive probes in Hela cells revealed the cytoplasm and nucleus had a pH of ≈7.3, mitochondria ≈8.0, ER ≈7.45 and Golgi ≈6.6 (BNID 105939, 105940, 105942, 105943). Even though hydrogen ions are ubiquitous in the exercises sections of textbooks, their actual abundance inside cells is extremely small. To see this, consider how many ions are in a bacterium or mitochondrion of volume 1 m3 at pH 7. Using the rule of thumb that 1 nM corresponds to Figure 1: Measuring the pH of cells in vivo using pH sensitive fluorescent protein. Adapted from Rick Orij, Microbiology, 155, 268–278, (2009). ≈1 molecule per bacterial cell volume, and recognizing that pH7 corresponds to a concentration of 10-7 M (or 100 nM), this means that there are about 100 hydrogen ions per bacterial cell at the typical pHs found in such cells as worked out in more detail in the calculation shown in Figure 2. This should be contrasted with the fact that there are in excess of a million proteins in that same cellular volume, each one containing several ionizable groups each of which has a pKa close to 7 and thus the tendency to gain or release a hydrogen ion. How can so many reactions involving hydronium ions work with so few ions in the cell ? On average, how long does an active site need to wait to find a charge required for a reaction? It is important to note two key facts: (i) cells have a strong buffering capacity as a result of metabolites and amino acid side chains and (ii) the hopping time of charges between different water molecules is very short in comparison with the reaction times of interest. We begin by discussing buffering. If the 100 hydrogen ions we have estimated are present in each cell were all used up to alter the charge state of macromolecules, the pH still does not change significantly as there are literally millions of groups in proteins and metabolites such as ATP that will compensate by releasing ions as soon as the pH begins to change. Hence, these 100 ions are quickly replenished whenever they are consumed in reactions. This implies that there are orders of magnitude more ion utilizing reactions that can take place. This capability is quantified by the cell’s buffering capacity. But how does a reaction “find” the hydronium ion to react with if they are so scarce? The lifetime of a hydronium ion is extremely brief, about 1 picosecond (10 -12, BNID 106548). Lifetime in this context refers to the “hopping” timescale when the charge will move to another adjacent water molecule (also called the Grotthuss mechanism). The overall effective diffusion rate is very high ≈7000 m2/s (BNID 106702). The lifetime and diffusion values can be interpreted to mean that for every ion present in the cell, on average, 1012 water molecules become charged very briefly every second. In an E. coli cell there are about 1011 water molecules. Thus every single ion “visits” every molecule 10 times a second and for 100 ions per cell every molecule will be converted to an ion 103 times per second, even if very briefly in each such case. As a result, an enzyme or reaction that requires such an ion will find plenty of them in the surrounding water Figure 2: back of the envelope calculation of the number of hydrogen ions in a cell volume. assuming the kinetics is fast enough to allow utilization before the ions neutralize to be formed somewhere else in the cell. The scale of the challenge of keeping a relatively constant pH can be appreciated by thinking of the first steps of sugar utilization. As glucose is catabolized in the process of glycolysis, internal electron rearrangements known as substrate-level phosphorylation, convert noncharged groups into a carboxyl acid group which releases a hydrogen ion. Consuming over 106 such glucose molecules per second the pH of the cell would rapidly change if not for the buffering effects and active regulation just discussed. This is but one example that illustrates the powerful and tightly-regulated chemistry of hydrogen ions in the molecular inventory of a cell.