Figure S1. Sequence alignment of yeast and horse cyt-c (Identity~60%), green highly conserved residues. There are 40 amino acid differences in the primary sequence and 5-additional at the N- terminal of yeast. 64 residues are invariant in yeast and horse plus the CXXCH -heme binding motif and Met-80. Supplementary Figures

pBTR1(WT) 5560 bp # # A Figure S2. Vector map of pBTR1 (unmodified) BamHI sites are marked by #

Figure S3B. Vector map of pBTR1 SU (SU – Shah Ubaid-ullah) modified having incorporated restriction sites MluI and XhoI at the start and end of CYC1 gene marked by *. BamHI sites are marked by #. Figure S3A. Cloning strategy adapted to insert restriction sites at the start and at the end of CYC1 gene. Fragment AB was amplified separately using primes Ub-1 and Ub-3 and CD was amplified using primers Ub-2 and Ub-4.Amplified fragments AB and CD were fused by denaturation and used as template in subsequent primer extension reaction using primers Ub-1 and Ub-4. Primers Ub-9 to Ub-5 carried MluI site and were used to delete the N-terminal residues of y-cyt-c. BamHI Ub-1 Ub-2 Ub-3 Ub-4 MluI XhoI Ub4 Ub-8 Ub-9 Ub-7 Ub-5 Ub-6 CYC1 AB C D Ub-1 Ub-4 # # pBTR1 SU 5544 bp * *

Figure S4. SDS PAGE of yeast iso-1-cyt c at different steps of purification. Lane 1: TCP (Total Cell Pellet), Lane 2: Supernatant (after cell lysis), Lane 3: Supernatant after NH 2 SO 4 Cut, Lane 4: SDS VI Marker, Lane 5: WT y-cyt-c, Lane 6:  (-5/-5), Lane 7:  (-5/-4), Lane 8:  (-5/-3), Lane 9:  (-5/-2), Lane 10:  (-5/-1).

Primer NoSequenceRestriction Site Ub-1 5'- TCG TAT GTT GTG TGG AAT TGT GAG CGG ATA -3' Ub-25’-CTT TAA GAA GGA GAT acg cgt ATG ACT GAA TTC AAG -3'MluI Ub-35’-CTT GAA TTC AGT CAT acg cgt ATC TCC TTC TTA AAG -3'MluI Ub-45'- ATA CTA TAg gat ccG ctc gag TTT ACT CAC TGG CTT -3'XhoI & BamH1 Table S1. Primers used for insertion of restriction sites in pBTR1 Table S2. Primers used for creating the N-terminal variants of yeast iso-1-cyt-c PrimersSequenceCyt-c Variant UB-95’-AAT ATA CTA acg cgt ATG GAA TTC AAG GCC GGT- 3’Δ(-5/-5) UB-85’ -AAT ATA CTA acg cgt ATG TTC AAG GCC GGT TCT- 3’Δ(-5/-2) UB-75’-AAT ATA CTA acg cgt ATG AAG GCC GGCT TCT GCT- 3’Δ(-5/-3) UB-65’-AAT ATA CTA acg cgt ATG GCC GGT TCT GCT AAG- 3’Δ(-5/-4) UB-55-’AAT ATA CTA acg cgt ATG GGT TCT GCT AAG AA- 3’Δ(-5/-1)