Evidence for [2Fe-2S]²⁺ and Linear [3Fe-4S]¹⁺ Clusters in a Unique Family of Glycine/Cysteine-Rich Fe-S Proteins from Megavirinae Giant Viruses

Abstract

We have discovered a protein with an amino acid composition exceptionally rich in glycine and cysteine residues in the giant virus mimivirus. This small 6 kDa protein is among the most abundant proteins in the icosahedral 0.75 μm viral particles; it has no predicted function but is probably essential for infection. The aerobically purified red-brownish protein overproduced in Escherichia coli contained both iron and inorganic sulfide. UV/vis, EPR, and Mossbauer studies revealed that the viral protein, coined GciS, accommodated two distinct Fe-S clusters: a diamagnetic S = 0 [2Fe-2S] 2+ cluster and a paramagnetic S = 5/2 linear [3Fe-4S] 1+ cluster, a geometry rarely stabilized in native proteins. Orthologs of mimivirus GciS were identified within all clades of Megavirinae, a Mimiviridae subfamily infecting Acanthamoeba, including the distantly related tupanviruses, and displayed the same spectroscopic features. Thus, these glycine/cysteine-rich proteins form a new family of viral Fe-S proteins sharing unique Fe-S cluster binding properties.

Multimodal Spectroscopic Analysis of the Fe–S Clusters of the as-Isolated Escherichia coli SufBC2D Complex

“…It displays a brown-blackish color with an unusual UV–vis absorption spectrum: a main absorption band at 420 nm and shoulders at 320, 530, and 620 nm (Figure B). This UV–vis spectrum is reminiscent of that described for a linear [3Fe-4S] cluster, with absorptions bands at 320, 415, 513, and 573 nm. − These results are somewhat different (Fe, S, and flavin contents; complex homogeneity) to those described by Saini et al that purified anaerobically the complex, and they might result to the different method/protocol used.…”

“…This signal likely corresponds to adventitiously bound high-spin ( S = 5/2) Fe 3+ species. No significant signal was visible at lower fields (Figure S3A), in particular around g = 9.6, where additional features are usually detected in the spectrum of linear [3Fe-4S] 1+ . ,, Comparison with a known sample containing a linear [3Fe-4S] 1+ cluster revealed a maximum possible concentration of 7.2 μM (Table and Figure S4B). This corresponds to 5.3% of iron versus the total iron content.…”

The Cryptic Nature of Fe-S Clusters: A Case Study of the Hepatitis B HBx Oncoprotein

“…Linear [3Fe-4S] 1+ clusters have a ground spin state of S = 5/2 and are characterized by a signal at g~9, along with additional resonances at 4.3 and 4.15 that are often detected up to 40 K [81,82]. Although they were initially produced from the cuboidal forms of trinuclear clusters either by partial unfolding or oxidative conditions, they have recently been identified in the GciS, a protein in the viral capsid of giant viruses [25]. The one-electron reduced [3Fe-4S] 0 state is also paramagnetic but has an integer spin ground state (S = 2), rendering it EPR-silent [1].…”

“…These are the catalytic subunit Nsp12 of the SARS-CoV-2 RNA-dependent polymerase [22], the Nsp13 helicase that associates with the RNA polymerase to afford replication of the viral genome [21], and the oncogenic protein sT of MCPyV that enhances DNA replication [24]. A very recent example is the low molecular weight protein GciS, which is the most abundant protein in the viral particle of Megavirinae giant viruses that coordinates a Fe-S cluster, for which the function is presently unknown [25]. The occurrence of Fe-S clusters in the viral proteome appears far more common than initially considered, and it is provoking to think that the incorporation of these cofactors must rely on the host Fe-S biosynthesis machinery, which is hoaxed into delivering these metalloclusters to viral recipients.…”

“…The Oncogenic Small Tumor Antigen of the Merkel Cell Polyomavirus (MCPyV) was also thought to coordinate two Zn 2+ ions, which were later spectroscopically demonstrated to be [2Fe-2S] and [4Fe-4S] clusters, respectively [24], instead. Fe-S clusters have also been discovered in the small protein R633b from the Megavirinae giant viruses, albeit with an unrecognized role [25].…”