Michael G. Finnegan

Variable temperature magnetic circular dichroism studies of reduced nitrogenase iron proteins and [4Fe-4S]+ synthetic analog clusters

Variable temperature magnetic circular dichroism (VTMCD) and EPR spectroscopies have been used to investigate the ground and excited-state properties of [4Fe-4S]+ clusters in Mo- and V-nitrogenase Fe-proteins from Azotobacter vinelandii and two synthetic analog clusters, [Fe4S4(SEt)4]3- and [Fe4S4(SC6H11)4]3-. The results indicate similar [4Fe-4S]+ clusters with analogous S = 1/2 and S = 3/2 ground states in both Fe-proteins. However, the Fe-proteins do differ in terms of the medium effects on the S = 1/2 and S = 3/2 spin mixtures in frozen solution. By utilizing medium effects in both Fe-proteins, the VTMCD characteristics of both the S = 1/2 and S = 3/2 forms of the [4Fe-4S]+ have been determined. Together with the VTMCD studies of [Fe4S4(SEt)4]3- and [Fe4S4(SC6H11)4]3-, which are shown to be predominantly S = 1/2 and 3/2, respectively, in frozen DMF/toluene solutions, the results demonstrate that the form of the VTMCD spectra provides a means of identifying and distinguishing S = 1/2 and S = 3/2 [4Fe-4S]+ clusters. Ground state zero-field splitting parameters for the S = 3/2 clusters are determined for both Fe-proteins. In addition to spin state heterogeneity, samples of the Mo-nitrogenase Fe-protein in the presence of 50% (v/v) ethylene glycol were found to exhibit heterogeneity in the S = 1/2 resonance. A rapidly relaxing axial resonance, g perpendicular = 1.94 and g parallel = 1.82, was observed in addition to the characteristic rhombic resonance, g = 2.05, 1.94 and 1.87. The origin of the heterogeneity exhibited by [4Fe-4S]+ clusters in frozen solution is discussed in light of these results.

Spectroscopic identification of the heme axial ligation of cytochrome b558 in the NADPH oxidase of porcine neutrophils

The combination of electron paramagnetic resonance (EPR), near‐infrared magnetic circular dichroism (NIR‐MCD) and resonance Raman (RR) spectroscopies at cryogenic temperatures has been used to identify the axial heme ligation of the low spin cytochrome b 558 component of NADPH oxidase from porcine blood neutrophils. The EPR and NIR‐MCD results indicate the presence of two distinct forms in frozen solution; one with a low field g‐value at 3.23 and porphyrin(π)‐to‐Fe(III) charge transfer maximum at 1660 nm and the other a low field g‐value at 3.00 and porphyrin(π)‐to‐Fe(III) charge transfer maximum at 1510 nm. On the basis of these properties and the RR studies, both are attributed to forms of cytochrome b 558 with bis‐histidine axial ligation. The origin of the observed heterogeneity, the location and identity of the specific histidines involved in ligating the heme, and the role of the heme prosthetic group in O2 − production are discussed in light of these results.

Axial heme ligation in the cytochrome bc1 complexes of mitochondrial and photosynthetic membranes. A near-infrared magnetic circular dichroism and electron paramagnetic resonance study

The combination of EPR and low-temperature near-IR magnetic circular dichroism spectroscopies have been used to investigate the axial ligation of the cytochromes in the cytochrome bc1 complexes from bovine heart mitochondria, Rhodobacter capsulatus, Rhodobacter sphaeroides, and Rhodospirillum rubrum, and the purified cytochromes c1 from bovine heart mitochondria, Rb. capsulatus and Rb. sphaeroides. The possibility of axial ligation of cytochrome c1 by the amino terminus of the polypeptide was also assessed by acetylating the N-terminus of Rb. capsulatus cytochrome c1 and comparing the properties of the acetylated and unmodified samples. The results are consistent with bis-histidine axial ligation for the high- and low-potential b-type cytochromes and histidine/methionine axial ligation for the c1-type cytochrome in the intact cytochrome bc1 complexes. Purified samples of cytochrome c1 are mixtures of two forms, one with histidine/methionine and the other with bis-histidine axial ligation. The form with bis-histidine axial ligation is also assembled in the M183L mutant of the Rb. capsulatus cyt bc1 complex in which the methionine residue coordinating cyt c1 is replaced by a leucine. The bis-histidine form appears to be an artifact of dissociation of cytochrome c1 from the cytochrome bc1 complex and is greatly enhanced particularly in the bacterial cytochromes c1 by sample handling and the addition of 50% (v/v) ethylene glycol or glycerol.