Induced fit on heme binding to the Pseudomonas aeruginosa cytoplasmic protein (PhuS) drives interaction with heme oxygenase (HemO).

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.

Identification of two heme-binding sites in the cytoplasmic heme-trafficking protein PhuS from Pseudomonas aeruginosa and their relevance to function.

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.

Catalytic turnover dependent modification of the Pseudomonas aeruginosa heme oxygenase (pa-HO) by 5,6-O-isopropyledine-2-O-allyl-ascorbic acid.

Heme oxygenase (HO) catalyzes the NADPH dependent conversion of heme to biliverdin with the release of iron and CO via three successive oxygenation steps. The oxidation of heme in the presence of alternate reductants, such as ascorbic acid, has been used extensively to characterize the mechanism of oxygen activation in HO without altering the chemistry of the reaction. NADPH-dependent cytochrome P450 reductase (CPR) and ascorbic acid mediated reactions are mechanistically very similar, in that both use molecular oxygen to initiate the reaction.

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

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.

P(450)/NADPH/O(2)- and P(450)/PhIO-catalyzed N-dealkylations are mechanistically distinct.

A high-valent iron-oxo species analogous to the compound I of peroxidases has been thought to be the activated oxygen species in P450-catalyzed reactions. Spectroscopic characterization of the catalytically competent iron-oxo species in iodosobenzene (PhIO)-supported model reactions and parallels between these model reactions and PhIO- and NADPH/O2-supported P450 reactions have been taken as strong evidence for this proposal. To support this proposal, subtle differences observed in regio- and chemoselectivities, isotope effects, and source of oxygen, etc.

Microsomal P450-catalyzed N-dealkylation of N,N-dialkylanilines: evidence for a C(alpha)-H abstraction mechanism.

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.