PhuS is a cytoplasmic, 39 kDa heme-binding protein from Pseudomonas aeruginosa. It has previously been shown to transfer heme to its cognate heme oxygenase. It is expressed from the phu operon, which encodes a group of proteins known to actively internalize and transport heme from host organisms. This study combines the spectral resolution of resonance Raman spectroscopy with site-directed mutagenesis to identify and characterize the heme-bound states of holo-PhuS. This combined approach has identified a site in monomeric PhuS having alternate His ligands at positions 209 and 212. A second distinct binding site is present in dimeric PhuS. This site supports six-coordinate, low-spin heme, even when both His209 and His212 are mutated to Ala. The presence of conserved His and Tyr residues in all of the homologs characterized to date suggest that the dimer could be of the domain-swapped type in which two protein molecules are cross-linked by bound heme. The multiple heme-bound states and their sensitivity to pH suggest the possibility that these cytoplasmic heme-binding proteins have multiple functions that are toggled by variations in intracellular conditions.

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 ironregulated heme oxygenase (pa-HO). While the heme association rate for PhuS was similar to that of myoglobin, a markedly higher rate of heme dissociation (∼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 (k H =1.3 × 10 −8 μM) than that of PhuS (0.2 μM). 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. Based on 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 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 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.Heme, a cofactor of proteins involved in a variety of biological processes such as oxygen transport and storage, oxygenation reactions, electron transfer and transcriptional regulation is also a redox-reactive, hydrophobic iron chelate that readily associates with membranes, and is toxic to cells due to its ability to generate reactive oxygen species. Therefore, aerobic organisms have developed strategies to protect themselves from the harmful effects of "free" heme by sequestering it within specific proteins (1,2). While hemeproteins se...

Iron, an essential nutrient with limited bioavailability, requires specialized cellular mechanisms for uptake. Although iron uptake into the cytoplasm in the form of heme has been well characterized in many bacteria, the subsequent trafficking is poorly understood. The cytoplasmic heme-binding proteins belong to a structurally related family thought to have evolved as “induced fit” ligand-binding macromolecules. One member, Pseudomonas aeruginosa cytoplasmic protein (PhuS), has previously been shown to be important for delivering heme to the iron regulated heme oxygenase (HemO). Spectroscopic investigations of the holo-PhuS complex revealed a dynamic heme environment with overlapping but distinct heme-binding sites with alternative coordinating heme ligands, His-209 or His-212. In the present work we establish a mechanism for how heme is transferred from PhuS to its partner, HemO. Using surface plasmon resonance and isothermal titration calorimetry, we have discovered that holo-PhuS, but not apo-PhuS, forms a 1:1 complex with HemO. Sedimentation velocity and limited proteolysis experiments suggest that heme binding to PhuS induces a conformational rearrangement that drives the protein interaction with HemO. Hydrodynamic analysis reveals that the holo-PhuS displays a more expanded hydrodynamic envelope compared with apo-PhuS, and we propose that this conformational change drives the interaction with HemO. We further demonstrate that replacement of His-212 by Ala disrupts the interaction of holo-PhuS with HemO; in contrast, the His-209-Ala variant can still complex with HemO, albeit more weakly. Together, the present studies reveal a mechanism that couples a heme-dependent conformational switch in PhuS to protein–protein interaction, the subsequent free energy of which drives heme release to HemO.

The early proposal that P450-catalyzed N-dealkylation of N,N-dialkylamines proceeds through a single-electron-transfer (SET) mechanism was later challenged in favor of the C(alpha)-H abstraction mechanism. In the present study, a series of N-alkyl-N-cyclopropyl-p-chloroaniline probes have been used to examine whether the P450-catalyzed N-dealkylations proceed through a C(alpha)-H abstraction and/or a SET mechanism, using phenobarbital-induced rat liver microsomal P450 enzymes as a model system. While the findings are highly consistent with a C(alpha)-H abstraction mechanism, further experimental evidence may be necessary to completely rule out the SET mechanism.