Substitution of valine for histidine 265 in carbon monoxide dehydrogenase from Rhodospirillum rubrum affects activity and spectroscopic states.

In carbon monoxide dehydrogenase (CODH) from Rhodospirillum rubrum, histidine 265 was replaced with valine by site-directed mutagenesis of the cooS gene. The altered form of CODH (H265V) had a low nickel content and a dramatically reduced level of catalytic activity. Although treatment with NiCl2 and CoCl2 increased the activity of H265V CODH by severalfold, activity levels remained more than 1000-fold lower than that of wild-type CODH. Histidine 265 was not essential for the formation and stability of the Fe4S4 clusters. The Km and KD for CO as well as the KD for cyanide were relatively unchanged as a result of the amino acid substitution in CODH. The time-dependent reduction of the [Fe4S4]2+ clusters by CO occurred on a time scale of hours, suggesting that, as a consequence of the mutation, a rate-limiting step had been introduced prior to the transfer of electrons from CO to the cubanes in centers B and C. EPR spectra of H265V CODH lacked the gav = 1.86 and gav = 1.87 signals characteristic of reduced forms of the active site (center C) of wild-type CODH. This indicates that the electronic properties of center C have been modified possibly by the disruption or alteration of the ligand-mediated interaction between the nickel site and Fe4S4 chromophore.

Spectroelectrochemical characterization of the metal centers in carbon monoxide dehydrogenase (CODH) and nickel-deficient CODH from Rhodospirillum rubrum.

Carbon-monoxide dehydrogenase (CODH) from Rhodospirillum rubrum contains two metal centers: a Ni-X-[Fe4S4]2+/1+ cluster (C-center) that serves as the COoxidation site and a standard [Fe4S4]2+/1+ cluster (B-center) that mediates electron flow from the C-center to external electron acceptors. Four states of the C-center were previously identified in electron paramagnetic resonance (EPR) and Mössbauer studies. In this report, EPR-redox titrations demonstrate that the fully oxidized, diamagnetic form of the C-center (Cox) undergoes a one-electron reduction to the Cred1 state (gav = 1.87) with a midpoint potential of -110 mV. The reduction of Cox to Cred1 is shown to coincide with the reduction of an [Fe4S4]2+/1+ cluster in redox-titration experiments monitored by UV-visible spectroscopy. Nickel-deficient CODH, which is devoid of nickel yet contains both [Fe4S4]2+/1+ clusters, does not exhibit EPR-active states or reduced Fe4S4 clusters at potentials more positive than -350 mV.

In vitro synthesis of the iron-molybdenum cofactor of nitrogenase. Purification and characterization of NifB cofactor, the product of NIFB protein.

The requirement of NIFB activity for the biosynthesis of iron-molybdenum cofactor (FeMo-co) can be satisfied by the addition of the low molecular weight product of NIFB, termed NifB cofactor (NifB-co). NifB-co has been purified to homogeneity by a unique one-step method. Addition of NifB-co into the FeMo-co synthesis system generated nitrogenase activity of 27-32 nmol of ethylene formed/min/nmol of iron. Iron is the only metal detected in the NifB-co. NifB-co-dependent in vitro FeMo-co synthesis is absolutely dependent on the presence of molybdate, homocitrate and active NIFNE protein in the reaction mixture. The cofactor appears to be a small Fe-S cluster synthesized by NIFB, as a precursor of FeMo-co. NifB-co did not display any EPR signal at 4 K in 0-4000 gauss range. A solution of NifB-co is greenish-brown in color, similar to FeMo-co. NifB-co exhibits a broad absorbance between 400 and 700 nm with no distinctive peaks or shoulders. NifB-co is stable to repeated freeze-thaw cycles and is also stable in N-methylformamide, the solvent used for the isolation of FeMo-co. The NifB-co is stable to a 5-min heat treatment at 60 degrees C. The cofactor is extremely O2-labile, with half-life of less then 15 s in air.

Cutaneous metastases in patients with metastatic carcinoma: a retrospective study of 4020 patients.

BACKGROUND: Most previous studies have found that cutaneous metastases occur infrequently and are rarely present at the time the cancer is initially diagnosed. OBJECTIVE: We studied patients with metastatic cancer to determine the overall frequency of skin metastases, the frequency that these were the first sign of extranodal disease, and the clinical and histologic features of the cutaneous lesions. METHODS: A 10-year period of tumor registry files was searched for patients with metastatic carcinoma and melanoma. For patients with skin metastases, medical records and pathology reports were also examined. RESULTS: Of 4020 patients with metastatic disease, 420 (10%) had cutaneous metastases; in 306 of them the skin metastases were the first sign of extranodal metastatic Breast cancer and melanoma were the most common. Nodules were the most frequent clinical presentation, although inflammatory, cicatricial, and bullous lesions were also noted. Incisional metastases were common. Histologic findings most frequently revealed adenocarcinoma that was sometimes suggestive of the site of origin. After recognition of skin metastases, mean patient survival ranged from 1 to 34 months depending on tumor type. CONCLUSION: Cutaneous metastases are not uncommon and frequently are the first sign of extranodal metastatic disease, particularly in patients with melanoma, breast cancer, or mucosal cancers of the head and neck.

Skin involvement as the presenting sign of internal carcinoma. A retrospective study of 7316 cancer patients.

From tumor registry data of 7316 cancer patients, we found 367 cases (5.0%) with skin involvement. Skin involvement was present at the time of presentation in 92 patients (1.3%), only 26 of whom had remote metastases. Skin involvement was the first sign of cancer in 59 patients (0.8%); 22 had direct extension of their tumor into the skin, 20 had local metastases, and 17 had distal metastases. Direct invasion was most common with breast cancer and second most common with oral cavity cancer. Local metastases were also most frequently caused by breast cancer but occurred in surgical scars in three women with pelvic cancer and in perianal abscesses in one patient with rectal carcinoma as well. Except for metastases from unknown primary sites, distant metastases were rare as presenting signs, and their origins were widely distributed. Our data show that internal cancer uncommonly presents with skin involvement. Nevertheless, an index of suspicion should be maintained and biopsy performed, particularly for nonhealing ulcers, persistent indurated erythema, and unexplained skin nodules.