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The issue concerning several matters in hydrogenase research, namely the formal oxidation states of nickel in all its paramagnetic intermediate state, the structure of the [NiFe] cluster, including the type of the third bridging ligand between the metals and the functional role of the active center of the enzyme in the hydrogen conversion process is addressed. The various experimental and theoretical considerations indicate a plausible assignment of the g tensors to a specific site.

The carbon monoxide complex of [NiFe] hydrogenase from Desulfovibrio vulgaris Miyazaki F is characterized by X-ray crystallography and absorption and resonance Raman spectroscopy. Nine crystal structures of the of [NiFe] hydrogenase in the CO-bound and CO-liberated forms are determined at 1.2,-1.4 A resolution.

Several studies are conducted to explain the x-ray crystallographic structure of the R2F subunit present in the ribonucleotide reductase (RNR) of Corynebacterium ammoniagenes. Managanese is shown to play an extremely significant role in the discussed radical formation process.

A study to elucidate the mechanism of electron transfer between cytochrome c(sub 3) and [NiFe] hydrogenase, the transient complex formation by these proteins by means of nuclear magnetic resonance spectroscopy was conducted. The results revealed that the positive charges of Lys60, Lys72, Lys95, and Lys101 around heme 4 were important for the formation of the transient complex with [NiFe] hydrogenase in the initial stage of the cytochrome (sub 3) reduction.

The article presents a study on hydrogenases. It mentions that the group of metalloenzymes catalyze dihydrogen conversion into electrons and protons as well as its reverse reaction. It also notes their provision of energy for organisms through molecular hydrogen oxidation along with balancing cell redox potential.

The interaction of ferriheme proteins with nitrite has recently attracted interest as a source for NO or other nitrogen oxides in mammalian physiology. However, met-hemoglobin (metHb), which was suggested as a key player in this process, does not convert nitrite unless small amounts of NO are added in parallel. We have recently reported that, in contrast, nitrophorins (NPs) convert nitrite as the sole substrate to form NO even at pH 7.5. which is an unprecedented case among ferrihemes [He. C.. and Knipp. M. (2009) J. Am. Clien,. Soc. /31, 12042-I 2043j. NP5, which comprise a class of unique heme b proteins from the saliva of the bloodsucking insect Rhodnius prolixus, appear in a number of concomitant isoproteins. Herein, the first spectroscopic characterization of the initial complexes of the two isoproteins NP4 and NP7 with nitrite is presented and compared to the data reported for metHb and met-myoglobin (metMb). Because upon nitrite binding, NPs, in contrast to metHb and metMb, continue to react with nitrite, resonance Raman spectroscopy and continuous wave electron paramagnetic resonance spectroscopy were applied to frozen samples. As a result. the existence of two six-coordinate ferriheme low-spin complexes was established. Furthermore. X-ray crystallography of NP4 crystals soaked with nitrite revealed the formation ofan η1-N nitro complex, which is in contrast to the 9'-O-bound nitrite in metMb and metHb. Stopped-flow kinetic experiments show that although the ligand dissociation constants of NP4 and NP7 (15-190 M-1)are comparable to those of metHb and metMb. the rates of ligand binding and release are significantly slower. Moreover, not only the reaction kinetics but also electron paramagnetic resonance spectroscopy reveals notable differences between the two isoproteins. [ABSTRACT FROM AUTHOR]

Characterizes the carbon monoxide complex of [NiFe]hydrogenase from Desulfovibrio vulgaris Miyazaki F. Use of X-ray crystallography and absorption and resonance Raman spectroscopy; Determination of the nine crystals of hydrogenase; Observation of distinct changes in electron density distribution.

Iron-sulfur clusters form one of the largest and most diverse classes of enzyme cofactors in nature. They may serve as structural factors, form electron transfer chains between active sites and external redox partners, or form components of enzyme active sites. Their specific role is a consequence of the cluster type and the surrounding protein environment. The relative effects of these factors are not completely understood, and it is not yet possible to predict the properties of iron-sulfur clusters based on amino acid sequences or rationally tune their properties to generate proteins with new desirable functions. Here, we generate mutations in a [2Fe-2S] cluster protein, the TmHydC subunit of the trimeric [FeFe]-hydrogenase from Thermotoga maritima, to study the factors that affect its redox potential. Saturation mutagenesis of Val131 was used to tune the redox potential over a 135 mV range and revealed that cluster redox potential and electronic properties correlate with amino acid hydrophobicity and the ability to form hydrogen bonds to the cluster. Proline scanning mutagenesis between pairs of ligating cysteines was used to remove backbone amide hydrogen bonds to the cluster and decrease the redox potential by up to 132 mV, without large structural changes in most cases. However, substitution of Gly83 with proline caused a change of HydC to a [4Fe-4S] cluster protein with a redox potential of -526 mV. Together, these results confirm the importance of hydrogen bonding in tuning cluster redox potentials and demonstrate the versatility of iron-sulfur cluster protein folds at binding different types of clusters.

The nitrophorins (NPs) comprise a group of NO transporting ferriheme b proteins found in the saliva of the blood sucking insect Rhodnius prolixus . In contrast to other nitrophorins (NP1-4), the recently identified membrane binding isoform NP7 tends to form oligomers and precipitates at higher concentrations in solution. Hence, solid-state NMR (ssNMR) was employed as an alternative method to gain structural insights on the precipitated protein. We report the expression and purification of (13)C,(15)N isotopically labeled protein together with the first ssNMR characterization of NP7. Because the size of NP7 (21 kDa) still provides a challenge for ssNMR, the samples were reverse labeled with Lys and Val to reduce the number of crosspeaks in two-dimensional spectra. The two electronic spin states with S = 1/2 and S = 0 at the ferriheme iron were generated by the complexation with imidazole and NO, respectively. ssNMR spectra of both forms are well resolved, which allows for sequential resonance assignments of 22 residues. Importantly, the ssNMR spectra demonstrate that aggregation does not affect the protein fold. Comparison of the spectra of the two electronic spin states allows the determination of paramagnetically shifted cross peaks due to pseudocontact shifts, which assists the assignment of residues close to the heme center.