Isolation, Properties, and Crystallization of an Iron-Chlorin Protein from Aspergillus niger

Abstract

A green iron-chlorin protein was purified 160-fold from a lyophilized extract of Aspergillus niger by ion-exchange chromatography and gel filtration with a yield of 25%. The purified preparation appeared nearly homogeneous on sedimentation analysis and the sedimentation coefficient of the protein at infinite dilution was 13.4S. Its molecular weight was calculated to be 2.8--3.2 X 10(5) from sedimentation and gel filtration data. The ferric form of the protein had absorption maxima at 587.5 and 708 nm in the visible region and a Soret band at 404 nm. High-spin ESR signals of a rhombically distorted ferric iron-chlorin complex were observed at g = 6.5 and 5.3 together with unidentified, weaker signals at g = 4.3 and 2.0. The ferric form reacted readily with cyanide to give a complex showing absorption peaks at 422 and 632 nm and a shoulder at about 595 nm. When the protein combined with cyanide its high-spin ESR signals disappeared and low-spin signals at g = 1.88, 2.29, anous form having absorption maxima at 622 and about 410 nm. The rate of reduction by dithionite was slightly reduced by the presence of either nitrite or sulfite, and greatly accelerated by the presence of hydroxylamine. The reduced spectrum obtained in the presence of hydroxylamine had maxima at 620 and about 420 nm. The ferric cyanide complex did not show any spectral change on addition of dithionite. The green prosthetic group could be extracted with acidified acetone and the absorption maxima of the pyridine ferrihemochrome were at 413 and 599 nm. On removal of metal from the prosthetic group the characteristic spectrum of a chlorin was obtained. The absorption maxima of a solution of the chlorin in benzene were at 403, 501, 537, 576, 595, and 655 nm, the 655 nm band being strongest of those in the visible region. No significant amount of flavin was detected in the purified preparation. The iron-chlorin protein catalyzed methyl viologen-linked reduction of hydroxylamine and also that of nitrite at a slower rate under the same conditions, but not evidence that it reduced sulfite was obtained in the present study. The purified preparation also had high catalase [ec 1.11.1.6] activity. Crystalline material was obtained by gradual concentration of the purified preparation at about pH 6.