Multiple low spin forms of the cytochrome c ferrihemochrome. EPR spectra of various eukaryotic and prokaryotic cytochromes c.

Brautigan, David L.; Feinberg, Benjamin A.; Hoffman, Brian M.; Margoliash, E.; Preisach, J.; Blumberg, W. E.

doi:10.1016/s0021-9258(17)32756-4

Cited by 204 publications

(53 citation statements)

References 74 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…hm) value for D, and by numerically diagonalizing the energy matrix corresponding to eqn (33) as a function of the external magnetic field B, in order to obtain all intra-doublet energy differences that fit the resonance condition of eqn (40). The thus obtained effective g-values can be plotted vs. the rhombicity g as illustrated in Fig.…”

Section: C Rhombogramsmentioning

confidence: 99%

EPR spectroscopy as a probe of metal centres in biological systems

Hagen

2006

Dalton Trans.

View full text Add to dashboard Cite

Molecular paramagnetism pervades the bioinorganic chemistry of V, Mn, Fe, Co, Ni, Cu, Mo, W, and of a number of non-biological transition elements. To date we can look back at half a century of fruitful EPR studies on metalloproteins, and against this background evaluate the significance of modern EPR spectroscopy from the perspective of a biochemist, making a distinction between conventional continuous wave X-band spectroscopy as a reliable work horse with broad, established applicability even on crude preparations, vs. a diffuse set of "advanced EPR" technologies whose practical application typically calls for narrowly focused research hypotheses and very high quality samples. The type of knowledge on metalloproteins that is readily obtainable with EPR spectroscopy, is explained with illustrative examples, as is the relation between experimental complexity and the spin value of the system.

show abstract

Section: C Rhombogramsmentioning

confidence: 99%

EPR spectroscopy as a probe of metal centres in biological systems

Hagen

2006

Dalton Trans.

View full text Add to dashboard Cite

show abstract

“…To begin the simulations, the EPR parameters of cyanide-bound heme 1 were evaluated. EPR analysis of low-spin forms of ferricytochrome c hemes shows that they can give a range of EPR signals with g z = 3.05–3.4, g y = 2.05–2.25, and g x = 1.25–1.40 at neutral pH . More relevantly, cyanide-bound ferric horse cytochrome c gives EPR signals at g z = 3.45, g y = 1.89, and g x = 0.93 .…”

Section: Results and Analysismentioning

confidence: 99%

“…EPR analysis of lowspin forms of ferricytochrome c hemes shows that they can give a range of EPR signals with g z = 3.05−3.4, g y = 2.05−2.25, and g x = 1.25−1.40 at neutral pH. 42 More relevantly, cyanidebound ferric horse cytochrome c gives EPR signals at g z = 3.45, g y = 1.89, and g x = 0.93. 42 Taking this into consideration, we started our simulations with g z = 3.2, g y = 1.7, and g x = 0.93 for low-spin heme 1 to best fit the raw data (Figure 5C and Table 2).…”

Section: ■ Results and Analysismentioning

confidence: 99%

See 1 more Smart Citation

Elucidating Electron Storage and Distribution within the Pentaheme Scaffold of Cytochrome c Nitrite Reductase (NrfA)

et al. 2021

View full text Add to dashboard Cite

Cytochrome c nitrite reductases (CcNIR or NrfA) play important roles in the global nitrogen cycle by conserving the usable nitrogen in the soil. Here, the electron storage and distribution properties within the pentaheme scaffold of Geobacter lovleyi NrfA were investigated via electron paramagnetic resonance (EPR) spectroscopy coupled with chemical titration experiments. Initially, a chemical reduction method was established to sequentially add electrons to the fully oxidized protein, 1 equiv at a time. The step-by-step reduction of the hemes was then followed using ultraviolet−visible absorption and EPR spectroscopy. EPR spectral simulations were used to elucidate the sequence of heme reduction within the pentaheme scaffold of NrfA and identify the signals of all five hemes in the EPR spectra. Electrochemical experiments ascertain the reduction potentials for each heme, observed in a narrow range from +10 mV (heme 5) to −226 mV (heme 3) (vs the standard hydrogen electrode). On the basis of quantitative analysis and simulation of the EPR data, we demonstrate that hemes 4 and 5 are reduced first (before the active site heme 1) and serve the purpose of an electron storage unit within the protein. To probe the role of the central heme 3, an H108M NrfA variant was generated where the reduction potential of heme 3 is shifted positively (from −226 to +48 mV). The H108M mutation significantly impacts the distribution of electrons within the pentaheme scaffold and the reduction potentials of the hemes, reducing the catalytic activity of the enzyme to 1% compared to that of the wild type. We propose that this is due to heme 3's important role as an electron gateway in the wild-type enzyme.

show abstract

“…pH-dependent experiments are commonly carried out to elucidate protein behaviour, for instance with the attempt to reproduce a certain (or altered) physiological environment [17], or in applied research, to examine the performance of enzymes in sub-optimal conditions which might be necessary for industrial utilization [18]. Many heme proteins have been described to undergo an alkaline transition at high pH related to different deprotonation processes [19][20][21][22][23]. Here, we use the well-characterized myoglobin (Mb) as model heme protein to explore a number of experimental setups using different alkaline buffers together with a chosen set of cryoprotectants to investigate the influence of such conditions on the EPR spectra.…”

Section: Introductionmentioning

confidence: 99%

Pitfalls in Sample Preparation of Metalloproteins for Low-Temperature EPR: The Example of Alkaline Myoglobin

2021

View full text Add to dashboard Cite

Due to fast relaxation processes of transition metal ions, electron paramagnetic resonance (EPR) spectroscopy of metalloproteins needs to be performed at cryogenic temperatures. To avoid damaging the biological system upon freezing, a cryoprotectant is generally added to the sample as a glassing agent. Even though cryoprotectants are expected to be inert substances, evidences in literature show their non-innocent role in altering the shape of EPR spectra of proteins and biological objects in general. In this work we conduct a systematic study on the impact of several experimental factors—such as buffer composition, choice of cryoprotectant, pH and temperature—on the EPR spectrum of myoglobin, taken as a reference system for being a well-characterized heme-containing protein. We focus on high-pH buffers to induce and investigate the alkaline transition of ferric myoglobin (pKa ~ 8.9). A combined approach of continuous-wave EPR and UV–visible absorption spectroscopy shows that using particular pairs of buffers and cryoprotectants determines a considerable pH variation in the sample and that this effect is enhanced at cryogenic temperature. In addition, phase memory times were measured to evaluate the efficiency of different cryoprotectants and compared with spectral linewidths in continuous-wave EPR. Our findings suggest that among the selected cryoprotectants ethylene glycol is rather effective, even more than the widely used glycerol, without having unwanted effects.

show abstract

Multiple low spin forms of the cytochrome c ferrihemochrome. EPR spectra of various eukaryotic and prokaryotic cytochromes c.

Cited by 204 publications

References 74 publications

EPR spectroscopy as a probe of metal centres in biological systems

EPR spectroscopy as a probe of metal centres in biological systems

Elucidating Electron Storage and Distribution within the Pentaheme Scaffold of Cytochrome c Nitrite Reductase (NrfA)

Pitfalls in Sample Preparation of Metalloproteins for Low-Temperature EPR: The Example of Alkaline Myoglobin

Contact Info

Product

Resources

About