The mechanism of heme transfer from the cytoplasmic heme binding protein PhuS to the delta-regioselective heme oxygenase of Pseudomonas aeruginosa

Abstract

The opportunistic pathogen Pseudomonas aeruginosa has evolved two outer membrane receptor-mediated uptake systems (encoded by the phu and has operons) by which it can utilize the hosts heme and hemeproteins as a source of iron. PhuS is a cytoplasmic heme binding protein encoded within the phu operon and has previously been shown to function in the trafficking of heme to the iron-regulated heme oxygenase (pa-HO). While the heme association rate for PhuS was similar to that of myoglobin, a markedly higher rate of heme dissociation (approximately 10(5) s(-1)) was observed, in keeping with a function in heme-trafficking. Additionally, the transfer of heme from PhuS to pa-HO was shown to be specific and unidirectional when compared to transfer to the non-iron regulated heme oxygenase (BphO), in which heme distribution between the two proteins merely reflects their relative intrinsic affinities for heme. Furthermore, the rate of transfer of heme from holo-PhuS to pa-HO of 0.11 +/- 0.01 s(-1) is 30-fold faster than that to apo-myoglobin, despite the significant higher binding affinity of apo-myoglobin for heme (kH = 1.3 x 10(-8) microM) than that of PhuS (0.2 microM). This data suggests that heme transfer to pa-HO is independent of heme affinity and is consistent with temperature dependence studies which indicate the reaction is driven by a negative entropic contribution, typical of an ordered transition state, and supports the notion that heme transfer from PhuS to pa-HO is mediated via a specific protein-protein interaction. In addition, pH studies, and reactions conducted in the presence of cyanide, suggest the involvement of spin transition during the heme transfer process, whereby the heme undergoes spin change from 6-c LS to 6-c HS either in PhuS or pa-HO. On the basis of the magnitudes of the activation parameters obtained in the presence of cyanide, whereby both complexes are maintained in a 6-c LS state, and the biphasic kinetics of heme transfer from holo-PhuS to pa-HO-wt, supports the notion that the spin-state crossover occur within holo-PhuS prior to the heme transfer step. Alternatively, the lack of the biphasic kinetic with pa-HO-G125V, 6-c LS, and with comparable rate of heme transfer as pa-HO is supportive of a mechanism in which the spin-change could occur within pa-HO. The present data suggests either or both of the two pathways proposed for heme transfer may occur under the present experimental conditions. The dissection of which pathway is physiologically relevant is the focus of ongoing studies.

Scheme I

Figure 1

Time course of Heme-Transfer from PhuS to pa-HO, paHO-mutants and BphO. (—) pa-HO (Wt); (– –) pa-HO-N19K/F117Y(DM); (−·−) pa-HO-DM-K132A; (−··−) pa-HO-DM-K34N; and (····) BphO. Experiments were conducted with 10μM PhuS, 30μM pa-HO and 30μM BphO in 20mM Tris-HCl, pH 7.5, at 25 °C, and time course were measured at 405nm.

Figure 2

Time course of Heme-Transfer from PhuS to pa-HO as a function of pH. (– –) pH 6.5; (—) 7.5; (····) 8.5; (–··–) pa-HO-G125V, pH 7.5; and (–·–) pa-HO-Wt with 10mM KCN, pH 7.5. Experiments were conducted with 10μM PhuS, and 30μM pa-HO in 20mM Tris-HCl at the corresponding pH at 25 °C, and time course were measured at 405 (for pH 6.5 and 7.5) and 410nm (pH 8.5). Reaction conducted in the presence of KCN was monitored at 419nm.

Figure 3

Arrhenius Plot of heme transfer in the presence of 10μM PhuS and 30μM pa-HO in 20mM Tris, pH 7.5. (o) Fast phase (k₁) and (•) slow phase (k₂).

Figure 4

Heme transfer from pa-HO (Panel A) and PhuS (Panel B) to myoglobin. Reactions were conducted in 20mM Tris-HCl, pH 7.5, at 25°C with 2μM pa-HO or PhuS and 12μM myoglobin and the time course was measured at 408nm.