Kinetic Study of Ligand Binding and Conformational Changes in Inducible Nitric Oxide Synthase

Abstract

Nitric oxide synthases (NOSs) are heme enzymes that generate highly reactive nitric oxide from l-arginine (l-Arg) in a complex mechanism that is still only partially understood. We have studied carbon monoxide (CO) binding to the oxygenase domain of murine inducible NOS (iNOS) by using flash photolysis. The P420 and P450 forms of the enzyme, assigned to a protonated and unprotonated proximal cysteine, through which the heme is anchored to the protein, show markedly different CO rebinding properties. The data suggest that P420 has a widely open distal pocket that admits water. CO rebinding to the P450 form strongly depends on the presence of the substrate l-Arg, the intermediate N-hydroxy-l-arginine, and the cofactor tetrahydrobiopterin. After adding these small molecules to the enzyme solution, the CO kinetics change slowly over the hours. This process can be described as a relaxation from a fast rebinding, metastable species to a slowly rebinding, thermodynamically stable species, which we associate with the enzymatically active form. Our results allow us to determine kinetic parameters of l-Arg binding to the ferrous deoxy iNOS protein for the first time and also provide clues regarding the nature of structural differences between the two interconverting species.

Homodimerization Counteracts the Detrimental Effect of Nitrogenous Heme Ligands on the Enzymatic Activity of Acanthamoeba castellanii CYP51

“…, loss of P450-CO species) is accompanied by the generation of a deligated photoproduct species with a Soret band typical of a ferrous pentacoordinate high-spin (HS) heme with a proximal thiolate ligand . We have recently observed similar absorbance difference spectra for another thiolate-ligated heme enzyme, inducible nitric oxide synthase (iNOS) . CO recombination results in recovery of the P450-CO Soret band at ∼450 nm at the expense of the photoproduct Soret at ∼405 nm, so that the absorbance differences decay to zero.…”

“…The kinetic time traces (Figure G) extend to longer times than those of P450 and thus indicate slower CO recombination. A similar observation was made in iNOS and rationalized with the distal heme pocket opening up in the P450 → P420 transition, making it accessible to water . In monomeric AcCYP51, the kinetics are markedly nonexponential, suggesting pronounced conformational heterogeneity .…”

“…41 We have recently observed similar absorbance difference spectra for another thiolateligated heme enzyme, inducible nitric oxide synthase (iNOS). 42 CO recombination results in recovery of the P450-CO Soret band at ∼450 nm at the expense of the photoproduct Soret at ∼405 nm, so that the absorbance differences decay to zero. Minor absorbance differences in the range ∼415−435 nm, that is, in between the two predominant P450-CO difference bands, are due to a small fraction of P420-CO species that had formed in the time course of the experiment (∼1.5 h), as will be discussed below.…”

“…A similar observation was made in iNOS and rationalized with the distal heme pocket opening up in the P450 → P420 transition, making it accessible to water. 42 In monomeric AcCYP51, the kinetics are markedly nonexponential, suggesting pronounced conformational heterogeneity. 43 The absorbance difference spectra and two-dimensional coutour plot (Figure 1H,I) reveal that CO photodissociation generates a pentacoordinate HS heme with a Soret band centered at ∼432 nm.…”