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Hemeproteins can act as catalysts, oxygen carriers or electron conductors. The ferric/ferrous reduction potential E(m7) of iron in the center of the prosthetic group ranges from negative values for peroxidases to an extreme positive value for cytochrome a(3) with Hb and Mb in the middle [1]. Proteins exercise their influence on E(m7) in several ways: via substituents at the periphery of the chelate structure, via the proximal ligand, and via interaction with the surrounding medium, amino acid side chains, or polar solvents. Work on recombined proteins and 2,4-substituted free hemes documented that the first two effects are additive [2]. For the third effect, models of the dielectric media on a molecular level have been successfully applied [3-5]. E(m7) has also been empirically correlated to the degree of heme exposure to water [6-8]. The apoprotein/porphyrin and water/porphyrin interfaces are complementary since water molecules fill any empty space in the crevice and surround any pertinent part of heme outside the protein boundary. The present work links to this idea by a combination of statistical mechanics simulations and quantum mechanical calculations comparing heme in water with heme in an apolar environment. Our results show that polarization of the porphyrin pi-electron cloud by the field from water dipoles influences E(m7). The dominant effect of this and other determinates of iron electron availability is perturbations of delocalized electron density in the porphyrin chelate, reproduced by a model where the prosthetic group is treated as a disc of uniform electron density. The present work is also of interest since the interfacial energy constitutes the main barrier for heme-protein separation [9-11].

In heme-containing proteins the heme group is usually non-covalently bound in a pocket. Molecular dynamics (MD) simulations have been performed to estimate the barrier height for heme–protein separation. In simulations of myoglobin dissolved in water, a force has been applied to pull the heme out of the binding pocket. With forces above 0.5 nN, the heme group is easily pulled out of the pocket in times of the order of tens of picoseconds. With weaker forces, heme release becomes too slow to be monitored in an MD simulation covering a couple of hundred picoseconds. These results are consistent with a free energy barrier to heme release of about 100 kJ/mol. The results show that the main energetic change that occurs during the release is a conversion of heme/protein Lennard–Jones energy into heme/water Lennard–Jones energy. The release is essentially barrierless in energy indicating that the main part of the barrier is entropic.

Inhibitors that belong to the serine protease inhibitor or serpin family have reactive centers that constitute a mobile loop with P1-P1' residues acting as a bait for cognate protease. Current hypotheses are conflicting as to whether the native serpin-protease complex is a tetrahedral intermediate with an intact inhibitor or an acyl-enzyme complex with a cleaved inhibitor P1-P1' peptide bond. Here we show that the P1' residue of the plasminogen activator inhibitor type 1 mutant (P1' Cys) became more accessible to radiolabeling in complex with urokinase-type plasminogen activator (uPA) compared with its complex with catalytically inactive anhydro-uPA, indicating that complex formation with cognate protease leads to a conformational change whereby the P1' residue becomes more accessible. Analysis of chemically blocked NH2 termini of serpin-protease complexes revealed that the P1-P1' peptide bonds of three different serpins are cleaved in the native complex with their cognate protease. Complex formation and reactive center cleavage were found to be rapid and coordinated events suggesting that cleavage of the reactive center loop and the subsequent loop insertion induce the conformational changes required to lock the serpin-protease complex.

The temperature dependence of the coordination of CO to two hemoproteins, myoglobin (Mb) and leghemoglobin (LegHb), has been studied by absorption spectroscopy. All spectral changes in the electronic and infrared ranges are fully reversible between 8 and 88°C and indicate a more linear orientation of the distal ligand with increasing temperature. The characteristic energy for the above spectral changes is around 200 cm −1 or identical to the Fe-Imidazole (Im) mode in hemoproteins. Our results are also compared with recent models for the dynamics of CO adsorbed at the surfaces of transition metals. Based on these surface models we suggest that our observed temperature dependences stem from thermal excitations of the Fe-Im vibration. This implies a so far undocumented mechanism by which the Fe-proximal bond influences the binding and energy dissipation at the active site of hemoproteins.

Dicarboxidine is a low- or noncarcinogenic benzidine-type chromogenic substrate to peroxidases. The color formation is of zero order from the onset of the reaction with a rate proportional to the enzyme concentration. Substrate solution and absorbance are stable for at least 24 h, the latter after an early decrease of 4% in 10 min. The complexity of the benzidine reaction necessitates precautions against side reactions. These are analyzed and suitable assay conditions are presented. Dicarboxidine is a valuable chromogen for studies of a slow generation of hydrogen peroxide, a slowly reacting substituted hydroperoxide, or a low peroxidase activity such as in the enzyme-linked immunosorbent assay or other immunological tests.

Lactoperoxidase (LP) [1] is a remarkable enzyme, being an efficient scavenger of peroxides, an oxidant of usual peroxidase substrates and halide ions [2] while also catalyzing the disproportionation of dithionite [3]. Like other peroxidases LP has a low redox potential and a protoheme prosthetic group, but a mixed spin type spectrum and a strong heme–protein attachment single out LP as being unique. For this reason we have studied the CO stretch of LP · CO at pH 5–9. The CO stretch of carbonyl horseradish peroxidases (HRP) A and C is pH dependent in terms of number and positions of ir bands [4]. This was interpreted as indicating the presence of a titratable group near the active center influencing the carbonyl resonator at low pH by H-bonding to the oxygen atom in CO. The ir spectrum of LP · CO exhibits CO stretches at 1940, 1955, and 1961 cm− (Fig.1 Spectra at intermediate pH values omitted). The 1940 cm− band predominates, and its position is independent of pH and buffer species. The 1955 cm− band is intense at ph 5, gives a shoulder at pH 7, and has vanished at pH 9. The 1961 cm− band changes in the reverse manner. The low intensity and essentially pH-independent band at ∼1913 cm− may not be related to bound CO. The ir spectra of LP and HRP A, C have in common a) a pH-independent stretch at 1937 ± 3 cm− with a half-band width 12 cm−; b) a broader band, sensitive to protons or dipoles. Stretch a) is attributed to CO in a narrow, apolar environment, whereas in b) CO may dwell in a more open site. The balance between the 1955 and 1961 cm− stretches may depend upon a change in polarity due to a proton or, more likely, a polar group. The properties a) and b) seem to be unique to peroxidases, although at present there is no explanation of why stretch b) is located on opposite sides of a) in HRP and LP. The active site of lactoperoxidase may be different from that of other peroxidases, either lacking the distal base, or the ligand binding sites being rigid and preventing carbonyl interaction with the distal base [5]. The redox potentials of LP and HRP A, C (−180, −212, and −265 mV) and vCO (1940, 1938, and 1933 cm−) of the a) stretch follow the correlation found for other heme proteins [4].