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Human serum albumin (HSA), the most prominent protein in blood plasma, is able to bind a wide range of endogenous and exogenous compounds. Among the endogenous ligands, HSA is a significant transporter of heme, the heme-HSA complex being present in blood plasma. Drug binding to heme-HSA affects allosterically the heme affinity for HSA and vice versa. Heme-HSA, heme, and their complexes with ibuprofen have been characterized by electronic absorption, resonance Raman, and electron paramagnetic resonance (EPR) spectroscopy. Comparison of the results for the heme and heme-HSA systems has provided insight into the structural consequences on the heme pocket of ibuprofen binding. The pentacoordinate tyrosine-bound heme coordination of heme-HSA, observed in the absence of ibuprofen, becomes hexacoordinate low spin upon ibuprofen binding, and heme dissociates at increasing drug levels. The electronic absorption spectrum and nu(Fe-CO)/nu(CO) vibrational frequencies of the CO-heme-HSA-ibuprofen complex, together with the observation of a Fe-His Raman mode at 218 cm(-1) upon photolysis of the CO complex and the low spin EPR g values indicate that a His residue is one of the low spin axial ligands, the sixth ligand probably being Tyr161. The only His residue in the vicinity of the heme Fe atom is His146, 9 A distant in the absence of the drug. This indicates that drug binding to heme-HSA results in a significant rearrangement of the heme pocket, implying that the conformational adaptability of HSA involves more than the immediate vicinity of the drug binding site. As a whole, the present spectroscopic investigation supports the notion that HSA could be considered as the prototype of monomeric allosteric proteins.

Nitrite is an important metabolite in the physiological pathways of NO and other nitrogen oxides in both enzymatic and nonenzymatic reactions. The ferric heme b protein nitrophorin 4 (NP4) is capable of catalyzing nitrite disproportionation at neutral pH, producing NO. Here we attempt to resolve its disproportionation mechanism. Isothermal titration calorimetry of a gallium(III) derivative of NP4 demonstrates that the heme iron coordinates the first substrate nitrite. Contrary to previous low-temperature EPR measurements, which assigned the NP4-nitrite complex electronic configuration solely to a low-spin (S = 1/2) species, electronic absorption and resonance Raman spectroscopy presented here demonstrate that the NP4-NO2(-) cofactor exists in a high-spin/low-spin equilibrium of 7:3 which is in fast exchange in solution. Spin-state interchange is taken as evidence for dynamic NO2(-) coordination, with the high-spin configuration (S = 5/2) representing the reactive species. Subsequent kinetic measurements reveal that the dismutation reaction proceeds in two discrete steps and identify an {FeNO}(7) intermediate species. The first reaction step, generating the {FeNO}(7) intermediate, represents an oxygen atom transfer from the iron bound nitrite to a second nitrite molecule in the protein pocket. In the second step this intermediate reduces a third nitrite substrate yielding two NO molecules. A nearby aspartic acid residue side-chain transiently stores protons required for the reaction, which is crucial for NPs' function as nitrite dismutase.

A distinctive characteristic of the class III heme peroxidases of the plant peroxidase superfamily is the presence of a pentacoordinate quantum mechanically mixed-spin heme state resulting from the admixture of S = 5/2 and S = 3/2 states. This is not observed in class I or II peroxidases and, in fact, is a very rare heme spin state. The corresponding hexacoordinate quantum mechanically mixed-spin state is even more uncommon and has not been observed in heme proteins. The presence of the pentacoordinate form in the class III peroxidases suggested that they could be ideal candidates to display also the six-coordinate quantum mechanically mixed-spin state. With this possibility in mind, the benzohydroxamic acid complexes of the class III peroxidases horseradish isoenzyme C and A2 and soybean peroxidase are studied by electronic absorption, resonance Raman, and EPR spectroscopy at room and low temperatures. The results are compared with those obtained for Coprinus cinereus peroxidase which belongs to class II...