Proton histidine cycle

In the past 30 years, there have been many speculations as to how the enzyme pumps protons [6], Until very recently, two major proposals have been mainly discussed. One proposal, due to Wikstrbm [23], was based on the so-called histidine cycle. This mechanism postulated that one of the His ligands of Cub acts as a molecular arm that carries the pumped protons and releases them in pairs. The physical principles of energy coupling to redox chemistry in this scheme are not clear. The other proposal, due to Michel [24], was based on the assumption that heme a is the pump element. A somewhat similar proposal is due to Yoshikawa [25], who suggests that there is a third H-channel for proton translocation. 

M. Wikstrom, Mechanism of Proton Translocation by Cytochrome c Oxidase A New Four-stroke Histidine Cycle. Biochim. Biophys. Acta, 1458,188-198,2000. 

The mechanism for the lipase-catalyzed reaction of an acid derivative with a nucleophile (alcohol, amine, or thiol) is known as a serine hydrolase mechanism (Scheme 7.2). The active site of the enzyme is constituted by a catalytic triad (serine, aspartic, and histidine residues). The serine residue accepts the acyl group of the ester, leading to an acyl-enzyme activated intermediate. This acyl-enzyme intermediate reacts with the nucleophile, an amine or ammonia in this case, to yield the final amide product and leading to the free biocatalyst, which can enter again into the catalytic cycle. A histidine residue, activated by an aspartate side chain, is responsible for the proton transference necessary for the catalysis. Another important factor is that the oxyanion hole, formed by different residues, is able to stabilize the negatively charged oxygen present in both the transition state and the tetrahedral intermediate. 

The behaviour of the mutant enzymes where, for example, histidine-152 has been changed to alanine is compared with that of wild type enzymes.60 The 31P NMR chemical shift values and signal width for H152A mutant enzyme have shown the presence of two conformers open and closed forms of the enzyme that interconvert slowly on the NMR time scale. The tightness of the binding of the cofactor to the protein surface and its protonation state have been also discussed for intermediate Schiff bases in different steps of the catalytic cycle (Table 1). 

The next question is, where the protons go to in the active site during the catalytic cycle. For the base, there are too many possibilities to be certain. Groups near to the dinuclear cluster that can accept or exchange protons include the side chains of the amino acids arginine and histidine, thiolate ligands to the cluster, and peptide NH groups. 

Fig. 7. (A) The oxidation states of Mn in the various S-states. The model incorporates a histidine radical formation in the Sj-state with no oxidation of Mn in the Sj- Sa transition the model also accommodates the 1 0 1 2 proton-release pattern in the Kok cycle (B) a proposed topological model for the photosynthetic water-oxidizing Mn-complex based on XAS and EPR studies. Figure source (A) [adapted] and (B) Sauer, Yachandra, Britt and Klein (1992) The photosynthetic water oxidation complex studied by EPR and X-ray absorption spectroscopy. In VL Pecararo (ed) Manganese Redox Enzymes, pp 141-175. VCH Publ.

Reaction mechanism Based on the observation of reaction intermediates in the crystal structure and on quantum chemical calculations Einsle et al. [148] propose an outline of the first detailed reaction mechanism of the cytochrome c Nir from W. succinogenes. Nitrite reduction starts with a het-erolytic cleavage of the weak N-O bond, which is facilitated by a pronounced backbonding interaction between nitrite and the reduced active site iron. The protons come firom a highly conserved histidine and tyrosine. Elimination of one of both amino acids results in a significant reduced activity. Subsequently, two rapid one-electron reductions lead to a FeNO form and, by protonation, to a HNO adduct. A further two-electron two-proton step leads to hydroxylamine. The iron in the hydroxylamine complex is in the Fe(III) state [149], which is unusual compared to synthetic iron-hydroxylamine complexes where the iron is mainly in the Fe(II) state. Finally, it readily loses water to give the product, ammonia. This presumably dissociates firom the Fe(III) form of the active site, whose re-reduction closes the reaction cycle. 

Understanding of the proton translocation has been complicated by the need to address a steady-state circumstance rather than to consider static states. For example, in the oxidized state in the absence of reductant, the heme-copper binuclear center relaxes to a state that lies outside of the catalytic cycle. With proper consideration of the catalytic cycle according to Wikstrdm and Verkhovsky, Two protons each are pumped during the oxidative and reductive halves of the cycle. This important insight has yet to be carried to the molecular species involved, although an OH" ligand for Cub[II] has been considered, as well as earlier implication of a histidine liganded to Cub. ... 

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