In this study, we report experimental results that provide the first complete challenge of a proposed model for heme acquisition by Staphylococcus aureus via the Isd pathway first put forth by Mazmanian, S. K., Skaar, E. P., Gaspar, A. H., Humayun, M., Gornicki, P., Jelenska, J.

The bacterium Staphylococcus aureus is responsible for numerous hospital-acquired infections ranging from superficial wound lesions to more severe infections such as pneumonia, osteomyelitis and septicaemia and, in some cases, death. The Isd (iron-regulated surface determinant) proteins expressed by S. aureus and select other bacteria are anchored to the bacterial cell wall and membrane and are involved in extracting haem from haemoglobin as an iron source. Our knowledge of the overall haem-scavenging mechanism on the bacterial surface is limited. A detailed description of the haem-binding properties in the transport pathway is critical to our understanding of the mechanism for haem-iron scavenging in S. aureus. Our work involves using a combination of techniques to characterize both the dynamic and steady-state haem-binding properties of these proteins. UV-visible absorption and MCD (magnetic circular dichroism) spectroscopy provide diagnostic spectral data sensitive to the axial ligands, the spin state and oxidation state of the central haem-iron. Electrospray MS provides stoichiometric information on the numbers of haems bound, the effect of haem binding on the overall folding of each protein and kinetic information about the rate of haem binding. Together, these data allow us to address the outstanding questions regarding the mechanism of haem transport via the Isd protein chain in S. aureus.

Iron is a critically important nutrient for all species. Bacteria have evolved specialist survival systems to chelate and transport iron across the wall and membrane into the cytoplasm. One such system in the human pathogenic bacteria Staphylococcus aureus involves extracting heme from hemoglobin and then transporting the intact heme across the wall and membrane. The iron-regulated surface determinant (Isd) proteins act in concert to carry out the heme scavenging and subsequent transport. While details of the static heme-binding reaction are currently quite well known, little mechanistic data are available. In this paper, we describe detailed time-resolved mass spectral and magnetic circular dichroism spectral data recorded as heme is transferred unidirectionally from holo-IsdA to apo-IsdE via IsdC. The electrospray mass spectral data simultaneously monitor the concentrations of six protein species involved in the trans-wall transport of the extracted heme and show for the first time the mechanistic details of heme transfer that is key to the Staphylococcus aureus Isd heme-scavenging system. Bimolecular kinetic analysis of the ESI-mass spectral data shows that heme transfer from IsdA to IsdC is very fast, whereas the subsequent heme transfer from IsdC to IsdE is slower. Under limiting IsdC conditions, the IsdC intermediary cycles between heme-free and heme-containing forms until either all heme has been transferred from holo-IsdA or no further apo-IsdE is available. The data show that a unique role for IsdC is acting as the central cog-wheel that facilitates heme transfer from IsdA to IsdE.

Absorption and magnetic circular dichroism (MCD) spectra, together with electrospray ionization mass spectral (ESI-MS) data are reported for the first two proteins in the Isd sequence of proteins in Staphylococcus aureus. IsdH-NEAT domain 3 (IsdH-N3) and IsdB-NEAT domain 2 (IsdB-N2) are considered to be involved in heme transport following heme scavenging from the hemoglobin of the host. The ESI-MS data show that a single heme binds to each of these NEAT domains. The charge states of the native proteins indicate that there is minimal change in conformation when heme binds to the heme-free native protein. Acid denaturation releases the bound heme and results in protein that exhibits significantly higher charge states, which we associate with unfolding of the protein structure. MCD spectra of the heme-bound native proteins show that the heme-iron is in a high-spin state, which is similar to that in IsdC-N. Addition of cyanide results in a spectral envelope characteristic of low-spin ferric hemes. The lack of complete binding for IsdH-N3 suggests that there is considerable congestion in the heme-binding site region. Unusually, reduction to the ferrous heme results in spectral characteristics of six coordination of the ferrous heme. CO is shown to bind strongly to both heme bound proteins, resulting in six-coordinate bound hemes. The spectra following reduction most closely resemble spectra recorded for heme with histidine in the fifth position and methionine in the sixth position. We report a theoretical model calculated from the X-ray structure coordinates of IsdH-N3, in which the heme is coordinated to nearby histidine and methionine. We propose that this structure accounts for the spectroscopic properties of the protein with the ferrous heme.

INTRODUCTION TO THE MCD TECH-NIQUEMagnetic circular dichroism (MCD) spectroscopy developed from magnetic optical rotary dispersion (MORD) spectroscopy, a measure of the variation in the optical rotation of a substance with a change in the wavelength of plane polarized light [1]. The change to MCD spectroscopy was slow partly because it was easier to measure ORD and MORD spectra than CD and MCD spectra. However, the great value of the CD spectrum in structural analysis of both biomolecules and chiral inorganic complexes accelerated the switch as new instruments became available in the late 1960's [2]. The MCD spectrum also was easier to work with because of the direct connection of the MCD signal morphology and magnitude with readily computed state to state transition properties. This is because the group theory of the 1960's provided an interpretation of the sign and estimate of the relative magnitudes of particularly, transition metal complexes then being studied [3].The instrumental difficulties in measuring CD spectra were greatly reduced with the development of the acoustooptic modulator that allowed measurement in all regions ABSTRACT: This review of the application of the MCD spectroscopic technique to porphyrinoids aims to illustrate the features and unique information that can be obtained and used in the assignment of the optical absorption spectrum and also the electronic structures of porphyrins and phthalocyanines. By illustrating each of the major and readily available spectral features with simple compounds we hope this review will act to guide new users in formulating the questions that room temperature MCD measurements can answer. The review is in three parts: (i) a brief introduction to the instrumental and theoretical background with an emphasis on the parameters that can be readily extracted without computational or theoretical analyses. (ii) Illustration of each MCD spectral feature observed in room temperature spectra. The MCD spectra Zn-porphyrins and phthalocyanines, and chlorophyll a are described to provide models for measurements of new compounds. (iii) The use of "finger printing" to determine the oxidation and spin states of the central iron in heme proteins, and to identify the proximal (5th) and distal (6th) iron binding ligands from the MCD spectral envelope observed between 250 nm and 1000 nm. The value of this approach is immeasurable because through room temperature measurement of the MCD spectra from dilute solutions under conditions that allow the heme protein to be stabilized it is possible to identify accurately the proximal and distal ligands. Identifying the heme binding environment for heme proteins newly purified and for heme enzymes during their reaction cycle allows function to be considered long before X-ray structural analysis is available.