Bioenergetic Processes

 

Bioenergetics is the branch of biochemistry that focuses on how cells transform energy, often by producing, storing or consuming adenosine triphosphate (ATP). Bioenergetic processes, such as cellular respiration or photosynthesis, are essential to most aspects of cellular metabolism, therefore to life itself. Bioenergetic processes, such as cellular respiration or photosynthesis, are essential to most aspects of cellular metabolism, therefore to life itself.

Cellular Respiration 

Cellular respiration refers to a catabolic process that cells use to harvest energy from biomolecules. Here, a series of reactions break up macromolecules into their basic units, transforming the potential energy embedded in the chemical bonds into ATPs and the cofactor nicotinamide adenine dinucleotide (NAD+). Cellular respiration in most organisms takes place in the presence of oxygen (aerobic respiration), consisting of the following pathways:

Glycolysis (Embden–Meyerhof–Parnas (EMP) pathway)

Glycolysis is the almost universal pathway that converts glucose into pyruvate along with the formation of nicotinamide adenine dinucleotide (NADH) and adenosine triphosphate (ATP). It primarily occurs in the cytoplasm of the cell. Glycolysis takes place in 10 steps, five of which are in the preparatory phase and five are in the pay-off phase.

The EMP pathway is present in organisms from every branch of the bacteria, archaea, and eukarya. It is suggested that the EMP pathway evolved in an anaerobic, fermentative world. However, the pathway also functions efficiently as the basis for aerobic respiration of glucose. The differences between fermentation and respiration lie largely in the differing fates of the pyruvate produced.

Before glucose metabolism begins, it must be transported into the cell and phosphorylated. In E. coli, these two processes are intimately coupled such that the glucose is phosphorylated by the phosphotransferase system (PTS) as it passes into the cell. Since glucose-6-phosphate (G-6-P), like most if not all sugar phosphates, is toxic at high cellular concentrations, this transport process is tightly regulated. Transcription of the glucose-specific transporter gene, ptsG, is maximal only when cyclic adenosine monophosphate (cAMP) (signaling energy limitation) accumulates. Moreover, translation of ptsG messenger RNA (mRNA) is inhibited by the small RNA sgrS, which is produced when G-6-P accumulates. Thus, the import and concomitant phosphorylation to G-6-P is reduced whenever the demand for more energy is low or the concentration of G-6-P is dangerously high.

In the absence of a PtsG protein, other PTS-linked transporters, especially the mannose-specific transporter, ManXYZ, can also transport and phosphorylate glucose. However, ptsG mutants grow more slowly on glucose than on wild-type strains. Free glucose can also accumulate intracellularly from the degradation of glucose-containing oligosaccharides such as lactose or maltose. Entry of intracellular glucose into the EMP pathway occurs via a hexokinase encoded by the glk gene.

The next two steps in the EMP pathway prepare the G-6-P for cleavage into two triose phosphates. First, a reversible phosphoglucose isomerase (pgi gene) converts G-6-P to fructose-6-phosphate. A pgi mutant can still grow slowly on glucose by using other glycolytic pathways, but the EMP pathway is blocked in a pgi mutant. The resulting fructose-6-phosphate is further phosphorylated at the C1 position to fructose-1,6,-bisphosphate at the expense of adenosine triphosphate (ATP) by a phosphofructokinase encoded by pfkA. A second minor isozyme of phosphofructokinase encoded by pfkB allows slow growth of pfkA mutants. A potentially competing set of phosphatases that remove the C1 phosphate from fructose-1,6,-bisphosphate function during gluconeogenesis but are controlled during glycolysis by a variety of feedback mechanisms to prevent futile cycling.

The next reaction in the pathway is the cleavage of fructose-1,6-bisphosphate to two triose phosphates that gives the pathway its name (glycolysis = sugar breakage). This reversible reaction is carried out by fructose bisphosphate aldolase (fbaA gene) and yields dihydroxyacetone phosphate (DHAP) and glyceraldehyde phosphate (GAP) as products. A second, unrelated aldolase (fbaB gene) is made only during gluconeogenesis and thus plays no role in glycolysis. The two triose phosphates are freely interconvertible via triosephosphate isomerase (tpi gene). DHAP is a key substrate for lipid biosynthesis.

The next step is the oxidative phosphorylation of GAP to 1,3-diphosphoglyceric acid, a high-energy compound. The incorporation of inorganic phosphate by GAP dehydrogenase (gapA gene) is coupled to the reduction of NAD⁺ to NADH. Under aerobic conditions, this NADH is reoxidized using the respiratory chain to yield ATP. Under anaerobic conditions, this NADH is reoxidized by coupling to the reduction of products derived from pyruvate or other EMP pathway intermediates. The enzyme phosphoglycerate kinase (pgk gene) then phosphorylates adenosine diphosphate (ADP) to ATP at the expense of the C1 phosphate of 1,3-diphosphoglycerate. This is the first of two substrate-level phosphorylations where phosphate is transferred from a highly reactive substrate directly to ADP without the involvement of the membrane ATP synthase.

The next two steps rearrange the resulting 3-phosphoglycerate to the last high-energy intermediate of the pathway, phosphoenolpyruvate (PEP). First, the phosphate is transferred from the C3 position to the C2 position by a phosphoglycerate mutase. There are two evolutionarily unrelated isozymes, one of which (encoded by the gpmA gene) requires a 2,3-bisphosphoglycerate as a cofactor and the other (gpmM gene) does not.

It is worth noting that PEP is a branch point under both aerobic and anaerobic conditions. The carboxylation of PEP by PEP carboxylase (ppc gene) provides oxaloacetate, which condenses with the acetyl-CoA derived from pyruvate to form citrate for running both the tricarboxylic acid (TCA) cycle and glyoxylate shunt aerobically. During fermentation, this same oxaloacetate is an intermediate in the reductive (NAD regenerating) pathway to succinate. In addition, the PEP-derived oxaloacetate is used for the biosynthesis of glutamic acid even under anaerobic conditions.

The last reaction is a substrate-level phosphorylation of ADP to ATP at the expense of PEP to yield pyruvate. The two isozymes of pyruvate kinase (pykA and pykF genes) are activated by sugar phosphates and the product of the pykF gene shows positive cooperativity with respect to the substrate PEP, again tending to prevent accumulation of this phosphorylated intermediate and thus preventing the generation of more G-6-P via the PEP-dependent PtsG transport mechanism.

At the end of the EMP pathway, 1 mol of glucose is converted to 2 mol of pyruvate, which can be used for further catabolism or for biosynthesis. It also yields 2 mol of ATP and 2 mol of NADH.

Pyruvate Decarboxylation

The reduction of NAD+ into NADH from glycolysis disrupts the redox state and depletes the cellular NAD+ reserve. As a result, pyruvate is further oxidized to replenish the NAD+ reserve. 

In eukaryotes, NADH and pyruvate are transferred to the mitochondria where they are oxidized to NAD+ and acetyl coenzyme A (acetyl-CoA) together with carbon dioxide (CO2), respectively. Acetyl CoA is further oxidized in the Citric Acid Cycle. The oxidative decarboxylation of Pyruvate to form Acetyl-CoA is the link between Glycolysis and the Citric acid cycle. The reaction occurs in the mitochondrial matrix. The pyruvate derived from glucose by glycolysis is dehydrogenated to yield acetyl CoA and CO₂ by the enzyme pyruvate dehydrogenase complex (PDC). It is an irreversible oxidation process in which the carboxyl group is removed from pyruvate as a molecule of CO₂ and the two remaining carbons become the acetyl group of Acetyl-CoA. High activities of PDC  are found in cardiac muscle and kidney.

Pyruvate Dehydrogenase Complex

It is a large multienzyme composed of Pyruvate dehydrogenase or Pyruvate decarboxylase (E1), Dihydrolipoyl transacetylase (E₂) and Dihydrolipoyl dehydrogenase (E₃). The enzyme also consists of 5 coenzymes viz., Thiamine pyrophosphate (TPP), Lipoic acid (LA), Flavin adenine dinucleotide (FAD), Coenzyme A (CoA) and Nicotinamide adenine dinucleotide (NAD⁺). All these enzymes and coenzymes are organized into a cluster to keep the prosthetic groups close together, thus allowing the reaction intermediates to react quickly with each other. These 3 enzyme components associate by the noncovalent bond to form the pyruvate dehydrogenase complex when they are mixed at neutral pH in the absence of urea.The organization of the PDH complex is very similar to that of the enzyme complexes that catalyze the oxidation of α-ketoglutarate and the branched-chain α-keto acids. The enzyme carries out the five consecutive reactions in the decarboxylation and dehydrogenation of pyruvate.

1.    Pyruvate reacts with the bound thiamine pyrophosphate (TPP) of pyruvate dehydrogenase (E₁), undergoing decarboxylation to from hydroxyethyl derivative of thiazole ring of TPP.

2.    Pyruvate dehydrogenase transfers two electrons and the acetyl group from TPP to the oxidized form of the lipoyllysyl group of the core enzyme, dihydrolipoyl transacetylase (E₂), to form the acetyl thioester of the reduced lipoyl group.

3.    It is a transesterification process in which the —SH group of CoA replaces the—SH group of E₂ to yield acetyl-CoA and the fully reduced (dithiol) form of the lipoyl group.

4.    Dihydrolipoyl dehydrogenase (E₃) promotes transfer of two hydrogen atoms from the reduced lipoyl groups of E₂ to the FAD prosthetic group of E₃, restoring the oxidized form of the lipoyllysyl group of E₂.

5.    The reduced FADH₂ of E₃ transfers a hydride ion to NAD⁺, forming NADH. The enzyme complex is now ready for another catalytic cycle.

The Citric (Tricarboxylic) Acid Cycle also known as Krebs cycle after Hans Adolf Krebs, the 1953 Nobel laureate who identified the cycle. The TCA cycle occurs at the mitochondrial membrane in aerobic respiration. It uses acetyl-CoA generated from glycolysis and pyruvate oxidation or the breakdown of lipids and amino acid.  Krebs cycle produces precursors of some amino acids, CO2, NADH, the hydroquinone form of flavin adenine dinucleotide (FADH2), and guanosine triphosphate (GTP) or ATP from substrate-level phosphorylation. NADH and FADH2 are subsequently used to synthesize ATP in oxidative phosphorylation. In eukaryotes, the citric acid cycle takes place in the matrix of the mitochondria, just like the conversion of pyruvate to acetyl Co A. In prokaryotes, these steps both take place in the cytoplasm. The citric acid cycle is a closed loop; the last part of the pathway reforms the molecule used in the first step. The cycle includes eight major steps. First, acetyl CoA combines with oxaloacetate, a four-carbon molecule, losing the CoA group and forming the six-carbon molecule citrate. After citrate undergoes a rearrangement step, it undergoes an oxidation reaction, transferring electrons to NAD+ to form NADH and releasing a molecule of carbon dioxide. The five-carbon molecule left behind then undergoes a second, similar reaction, transferring electrons to NAD+ to form NADH and releasing a carbon dioxide molecule. The four-carbon molecule remaining then undergoes a series of transformations, in the course of which GDP and inorganic phosphate are converted into GTP—or, in some organisms, ADP and inorganic phosphate are converted into ATP—an FAD molecule is reduced to FADH2, and another NAD+ is reduced to NADH. At the end of this series of reactions, the four-carbon starting molecule, oxaloacetate, is regenerated, allowing the cycle to begin again. In the first step of the citric acid cycle, acetyl CoA joins with a four-carbon molecule, oxaloacetate, releasing  a six-carbon molecule called citrate. In the second step, citrate is converted into its isomer, isocitrate. This is actually a two-step process, involving first the removal and then the addition of a water molecule. In the third step, isocitrate is oxidized and releases a molecule of carbon dioxide, leaving behind a five-carbon molecule—α-ketoglutarate. During this step, NAD+ is reduced to form NADH. The enzyme catalyzing this step, isocitrate dehydrogenase, is important in regulating the speed of the citric acid cycle. The fourth step is similar to the third. In this case, it’s α-ketoglutarate that’s oxidized, reducing NAD+ to NADH and releasing a molecule of carbon dioxide in the process. The remaining four-carbon molecule picks up Coenzyme A, forming the unstable compound succinyl CoA. The enzyme catalyzing this step, α-ketoglutarate dehydrogenase, is also important in regulation of the citric acid cycle. Succinyl CoA is converted to succinate in a reaction catalyzed by the enzyme succinyl-CoA synthetase. This reaction converts inorganic phosphate, Pi, and GDP to GTP and also releases a SH-CoA group. Therafter,  Succinate is converted to fumarate in a reaction catalyzed by succinate dehydrogenase. FAD is reduced to FADH2 in this reaction. Fumarate is converted to malate in a reaction catalyzed by the enzyme fumarase. This reaction requires a water molecule as a reactant. Malate is converted to oxaloacetate in a reaction catalyzed by malate dehydrogenase. This reaction reduces an NAD+ molecule to NADH + H+.

Photosynthesis

Photosynthesis takes place when photoreceptors in chloroplasts capture light. The energy acquired from light excites electrons, resulting in charge separation and subsequent electron transport reactions, termed photophosphorylation. Photophosphorylation takes place at the thylakoid membrane of the chloroplast, where two protein complexes, Photosystem I (PSI) and Photosystem II (PSII), are located. After light energy is harvested, the excitation and transfer of electrons lead to the oxidation of water to O2 and reduction of nicotinamide adenine dinucleotide phosphate (NADP+) to NADPH at the PSII and PSI, respectively. Similar to oxidative phosphorylation, electron transfer in chloroplasts is coupled with the generation of proton gradients across the thylakoid membrane, which drives the generation of ATP by ATP synthase. Both NADPH and ATP are used in the Calvin cycle to generate starch.