Evidence for pH-dependent multiple conformers in iron(II) heme-human serum albumin: spectroscopic and kinetic investigation of carbon monoxide binding.

Human serum albumin (HSA), the most prominent protein in plasma, is best known for its exceptional ligand binding capacity. HSA participates in heme scavenging by binding the macrocycle at fatty acid site 1. In turn, heme endows HSA with globin-like reactivity and spectroscopic properties. A detailed pH-dependent kinetic and spectroscopic investigation of iron(II) heme-HSA and of its carbonylated form is reported here. Iron (II) heme-HSA is a mixture of a four-coordinate intermediate-spin species (predominant at pH 5.8 and 7.0), a five-coordinate high-spin form (mainly at pH 7.0), and a six-coordinate low-spin species (predominant at pH 10.0). The acidic-to-alkaline reversible transition reflects conformational changes leading to the coordination of the heme Fe(II) atom by the His146 residue via its nitrogen atom, both in the presence and in the absence of CO. The presence of several species accounts for the complex, multiexponential kinetics observed and reflects the very slow interconversion between the different species observed both for CO association to the free iron(II) heme-HSA and for CO dissociation from CO-iron(II) heme-HSA as a function of pH.